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THE ROLES OF METALLOTHIONEIN EXPRESSION AND DIETARY ZINC IN 

ZINC METABOLISM AND CYTOPROTECTION IN METALLOTHIONEIN 

TRANSGENIC AND METALLOTHIONEIN KNOCKOUT MICE 



By 
STEVEN ROGER DAVIS 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

2000 



This work is dedicated to the memory of my aunt, Leona Goguen. She has been, and will 
always be my role model. I dream that one day, I too will possess the wisdom, character, 
and courage that she displayed each day of her life. 



ACKNOWLEDGMENTS 

I would like to thank my committee chairman, Dr. Robert J. Cousins for his 
guidance throughout this work. 

I would like to thank my committee members Dr. Jesse Gregory, Dr. Rachel 
Shireman, Dr. Susan Percival, and Dr. Stephen Roberts for their advice and input into 
this work. 

I would like to thank Walter Jones, Virginia Mauldin and Warren Clark for their 
technical assistance and sense of humor throughout my stay in Dr. Cousins' laboratory. 

I would like to thank Dr. Nora Holquist, Dr. Christina Khoo, Dr. Barbara Davis, 
Dr. Vicki Sullivan, Dr. Lorraine Lanningham-Foster, Dr. Jay Cao, Monique Coy, 
Jennifer Moore and Juan Liuzzi for their friendship and support. 

I would like to thank Dr. Raymond Blanchard and Dr. Robert McMahon for their 
friendship and tutoring in the laboratory. 

I would like to thank my family for their patience during this process. 

Lastly, I would like to thank Amy Mackey for her technical and professional 
assistance and especially for her personal support during the final stages of this work. 



in 



TABLE OF CONTENTS 

page 

ACKNOWLEDGMENTS Hi 

ABBREVIATIONS vi 

ABSTRACT vii 

CHAPTERS 

1 INTRODUCTION 1 

Literature Review 2 

Hypotheses and Research Objectives 9 

2 THE EFFECT OF METALLOTHIONEIN EXPRESSION ON ZINC 

ABSORPTION IN METALLOTHIONEIN TRANSGENIC AND 

METALLOTHIONEIN KNOCKOUT MICE 11 

Introduction 11 

Materials and Methods 12 

Results 15 

Discussion 21 

3 REGULATION OF METALLOTHIONEIN EXPRESSION 

AND ZINC METABOLISM BY DIETARY ZINC IN METALLOTHIONEIN 

TRANSGENIC AND METALLOTHIONEIN KNOCKOUT MICE 28 

Introduction 28 

Materials and Methods 33 

Results 32 

Discussion 36 

4 THE EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND 

SUPPLEMENTAL DIETARY ZINC IN PROTECTION AGAINST 
HEPATOTOXICITY IN METALLOTHIONEIN TRANSGENIC AND 

METALLOTHIONEIN KNOCKOUT MICE 41 

Introduction 41 

Materials and Methods 43 

Results 47 

Discussion 56 



IV 



5 EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND 

SUPPLEMENTAL ZINC IN PROTECTION AGAINST OXIDATIVE 
STRESS IN PRIMARY HEPATOCYTE CULTURES FROM 
METALLOTHIONEIN TRANSGENIC AND METALLOTHIONEIN 

KNOCKOUT MICE 66 

Introduction 66 

Materials and Methods 67 

Results 72 

Discussion 76 

6 SUMMARY AND CONCLUSIONS 85 

Zinc and Metallothionein in Zinc Absorption and Metabolism 85 

Zinc and Metallothionein in Defense Against Oxidative Stress 89 

LITERATURE CITED 94 

BIOGRAPHICAL SKETCH 109 



ABBREVIATIONS 



AAS 

ALT 

BBM 

CK 

CO 

CT 

Dex 

DTNB 

GSH 

GSSG 

i.p. 

II- 1 

11-6 

KO 

LDH 

LPS 

MRE 

MT 

MTT 

NPT 

pv 

SPAEC 

SSA 

TBH 

TCA 

TG 

TT 

WME 



atomic absorption spectrophotometry 

alanine aminotransferase enzyme 

brush border membrane 

control mouse strain for KO mice 

corn oil 

control mouse strain for TG mice 

dexamethasone 

5,5'-dithio-bis(2-nitrobenzoic acid) 

reduced glutathione 

oxidized glutathione 

intraperitoneal 

interleukin- 1 

interleukin-6 

metallothionein-null (knockout) mouse 

lactate dehydrogenase enzyme 

lipopolysaccharide 

metal response element 

metallothionein 

thiazoyl blue 

nonprotein thiol 

perivenous 

sheep pulmonary artery endothelial cell 

sulfosalicylic acid 

tertiary-butyl hydroperoxide 

trichloroacetic acid 

metallothionein overexpressing transgenic mouse 

total thiol 

William's medium E 



VI 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfillment of the 

Requirements for the Degree of Master of Arts 

THE ROLES OF METALLOTHIONEIN EXPRESSION AND DIETARY ZINC IN 

ZINC METABOLISM AND CYTOPROTECTION IN METALLOTHIONEIN 

TRANSGENIC AND METALLOTHIONEIN KNOCKOUT MICE 

By 

Steven Roger Davis 

December 2000 

Chairman: Robert J. Cousins 

Major Department: Food Science and Human Nutrition Department 

The biochemistries of zinc and metallothionein are intricately linked. Zinc 

induces metallothionein gene expression and allows the protein to resist proteolysis. In 

turn, binding of zinc to metallothionein allows cellular zinc accumulation, and 

metallothionein may be involved in intracellular zinc trafficking. Further, metallothionein 

induction and zinc accumulation often are coupled. This relationship makes it difficult to 

determine if effects of zinc supplementation are due to zinc, metallothionein induction, or 

both. Metallothionein overexpressing (transgenic) mice and metallothionein null 

(knockout) mice provide unique models to study the effects of zinc supplementation and 

metallothionein gene expression on physiological processes. We used these mouse 

models to determine (1) the effects of metallothionein expression and dietary zinc intake 

on zinc absorption and tissue zinc accumulation, and (2) the effects of zinc 



vn 



supplementation and/or metal lothionein gene expression on susceptibility to oxidative 
stress. 

Metallothionein expression was inversely proportional to serum zinc 
concentrations 2 h after an oral zinc dose, which strengthens the theory that 
metallothionein impedes zinc absorption. Intestinal zinc accumulation was inversely 
related to metallothionein expression, however, which argues that metallothioneinein 
does not act by simply sequestering zinc in the intestine. Metallothionein protein 
expression was directly proportional to intestinal and liver zinc concentrations after 3-7 d 
of zinc supplementation, but only at dietary zinc levels 20- to 50-fold of the requirement. 
Intestine and liver zinc concentrations did not change over a wide range of zinc intakes, 
and knockout mice maintained serum zinc levels as well as control mice. These points 
suggest that at typical zinc intakes, and in the absence of significant stresses, 
maintenance of zinc homeostasis is not metallothionein-dependent. 

Metallothionein expression protected against carbon tetrachloride-induced 
hepatotoxicity. Neither zinc supplementation nor metallothionein overexpression 
provided further protection, however. The heightened toxicity in KO mice after carbon 
tetrachloride treatment was associated with their lack of control over zinc homeostasis. In 
primary hepatocyte cultures, metallothionein induction was associated with increased 
susceptibility to oxidative stress. This was likely due to the observed depression of 
cellular glutathione. These results argue against direct antioxidant roles for 
metallothionein expression and supplemental zinc in mouse liver. 



Vlll 



CHAPTER 1 
INTRODUCTION 



The objectives of the research reported in this dissertation were to determine (1) 
the roles that dietary zinc and metallothionein have in regulation of zinc absorption and 
tissue distribution, and (2) the roles that dietary zinc and metallothionein have in defense 
against oxidative stress. The production of mouse strains with perturbed metallothionein 
expression provided the opportunity to investigate the above objectives under conditions 
of metallothionein absence and metallothionein excess in an intact animal model. 
Previously, investigators studying the effects of metallothionein expression on zinc 
metabolism or oxidative stress used a myriad of treatments to alter metallothionein levels 
before their experiments. Although they successfully altered metallothionein levels, other 
physiological pathways may have been perturbed as well. For example, zinc pretreatment 
induces metallothionein, but undoubtedly affects other components of the machinery that 
regulates zinc metabolism (e.g., zinc transporter abundance). Similarly, treatment with 
other metals, hormones, and cytokines is known to affect more than metallothionein. By 
using mice with altered metallothionein expression the complications associated with 
such treatments were avoided. Because of the artificial nature of these models, we must 
use caution when interpreting the results of studies in which they were used. 
Nevertheless, the results of such studies provided strong support for existing theories, as 
well as new insights for putative roles for metallothionein. 



Literature Review 
Zinc 

Zinc is an essential nutrient whose recommended dietary allowance (RDA; 15 
mg/d for men, 12 mg/d for women) ranks with iron as highest among the trace elements 
(National Research Council 1989). Zinc functions in more than 50 enzymes, serving in 
catalytic, structural, and regulatory roles (reviewed by Vallee and Falchuck 1993, 
Cousins 1996). These enzymes are involved in the synthetic and catabolic pathways of 
many biomolecules, including proteins, nucleic acids, carbohydrates and lipids. Zinc also 
is a component of zinc finger transcription factors. Zinc is distributed throughout the 
body, including 57% residing in skeletal muscle, followed by 29% in bone, 6% in skin, 
and 5% in liver (reviewed by Jackson 1989). Within cells zinc is distributed ubiquitously. 
Thiers and Vallee (1957) reported that 43% of liver zinc is cytosolic, 37% nuclear, 13% 
microsomal, 5% mitochondrial, and 2% in connective tissue. Zinc deficiency is 
associated with a 20% reduction in whole body zinc, but it is unknown which 
intracellular pool(s) is most affected (Hambidge 1989). Although severe zinc deficiency 
is uncommon, mild zinc deficiency may be prevalent in many parts of the world (Prasad 
1991). 

Metallothionein 

Metallothioneins are a family of small (6-7 kDa), cysteine-rich metal binding 
proteins found in vertebrates and invertebrates (Dunn et al. 1987). These proteins are 
further characterized by a lack of histidine residues, aromatic amino acids, and disulfide 
bonds. The protein is capable of binding up to (10) copper atoms, or up to (7) cadmium 
and/or zinc atoms in two distinct clusters of the protein (Neilson et al. 1985). 






Metallothioneins are thought to function in metal homeostasis, including zinc absorption 
and tissue distribution, and protection against heavy metal toxicity (Cousins 1985, Liu et 
al. 1995, Masters et al. 1994A). Metallothioneins are transcriptionally regulated by 
metals through metal response elements (MREs) in their gene promoters (Carter et al. 
1984, Durnam and Palmiter 1981, Stuart et al. 1984). Metallothioneins also are 
transcriptionally regulated by glucocorticoid hormones and cytokines, which implies a 
role for metallothioneins in inflammatory and stress-related responses, such as the acute 
phase response (Cousins and Leinart 1988, Etzel et al. 1979). 

Although four forms of metallothionein have been discovered, the two most 
widely expressed are metallothionein- 1 and metallothionein-2 (Hunziker et al. 1995, 
Kagi et al. 1974). These forms are found in most tissues, and are especially prevalent in 
the liver, kidney, pancreas, and intestine (Hunziker and Kagi 1985). Metallothionein-3 
and metallothionein-4 forms were discovered recently, and are expressed mostly in the 
brain and skin, respectively (Masters et al. 1994B, Quaife et al. 1994). Subforms of 
metallothionein (e.g., MT-la) exist in primates, but not in mice (Stennard et al. 1994, 
Twunoo et al. 1978). Because metallothionein- 1 and metallothionein-2 are the 
predominant forms in the liver and the intestine, they are the focus of discussion from 
this point on. 

Metallothionein in Zinc Absorption and Tissue Distribution 

Chapters 2 and 3 of this dissertation focus on the role of metallothionein in 
dietary zinc absorption and tissue zinc accumulation. The quantity of zinc absorbed by 
the body depends on several processes. These include the ingestion and digestion of zinc- 
containing foodstuffs, uptake of zinc by the intestinal mucosa, and transport of zinc from 



the intestine to the vascular supply. Zinc uptake by the intestine, followed by transfer to 
the portal blood supply is referred to collectively as zinc absorption. Zinc is absorbed 
throughout the entire small intestine (Lee et al. 1989). The predominant site in humans is 
the jejunum, while both the duodenum and jejunum appear to be major sites in rodents 
(Davies 1980, Lee et al. 1989). Although some information is known about zinc 
absorption, a clear understanding of this process and its regulation has not been reached. 
The process can be divided into three parts: (1) uptake from the lumen at the brush 
border membrane (BBM), (2) transport across the epithelial cell, and (3) transfer of zinc 
across the basolateral membrane to the vascular bed. Zinc absorption is dependent on the 
concentration of bioavailable zinc in the lumen. Although ~ 30% of zinc is absorbed 
from typical diets, greater efficiency is achieved in animals fed zinc-deficient diets, and 
lesser efficiency in animals fed diets containing supplemental zinc (Hempe and Cousins 
1992, Sandstrom 1989, Smith et al. 1978). In vitro transport studies using brush border 
membrane vesicles from rats showed that BBM transport was greater from zinc-deficient 
rats compared to zinc-adequate rats (Menard and Cousins 1983). Dietary zinc intake did 
not affect transport into basolateral membrane vesicles, however, suggesting that control 
of zinc absorption occurs at the apical, but not the basolateral membrane (Oestreicher and 
Cousins 1989). 

The last area for control of zinc absorption is the enterocyte cytosol itself. The 
cytosolic protein metallothionein has been the focus of research in this area because it is 
induced by high dietary zinc and parenteral zinc administration, but is depressed at low 
zinc intakes (Menard and Cousins 1983, Smith et al. 1978). One model of zinc absorption 
suggests that intestinal metallothionein is an integral part of the regulatory machinery, 






acting as a damping agent during periods of excessively high dietary zinc intakes 
(Cousins 1989, Hoadley et al. 1988, Richards and Cousins 1975). In this model a high 
zinc influx into the mucosal cell induces metallothionein production. Metallothionein 
then chelates the cytosolic zinc, limiting its passage from the enterocyte to the portal 
circulation. Enterocyte to lumen efflux of zinc, combined with regular sloughing of 
enterocytes from the villus tip result in reduced zinc absorption. This model is consistent 
with results from animal studies wherein high zinc diets or parenteral zinc administration 
elevated metallothionein levels in the intestine and resulted in decreased zinc absorption 
from subsequent meals (Coppen and Davies 1987, Hoadley et al. 1988, Richards and 
Cousins 1975). Similarly dietary zinc restriction depresses tissue metallothionein content 
and results in enhanced zinc absorption from subsequent meals (Hoadley et al. 1987, 
Smith and Cousins 1980). 

Contrary to results with diet-related changes in metallothionein, several inducers 
of intestinal metallothionein do not inhibit zinc absorption. For instance, bacterial 
infection, bacterial lipopolysaccharide injection, and interleukin- 1 administration each 
increased zinc absorption, even though each induces intestinal metallothionein (Pekarek 
and Evans 1975, Pekarek and Evans 1976). Further, mouse studies showed no clear 
correlation between metallothionein level of the intestine and zinc absorption using either 
oral dosing, stomach tube feeding, in situ duodenal loop feeding, or injection of a zinc 
solution into the duodenum in situ (Flanagan et al. 1983, Olafson 1983, Starcher et al. 
1980). Other studies have shown no increase in intestinal zinc retention even when 
metallothionein is induced (Hempe et al. 1991). Thus the role of metallothionein in 
regulation or zinc absorption is still in question. 



Metallothionein also is believed to affect zinc accumulation in tissues. 
Metallothionein gene expression is regulated in a tissue-specific manner in rats (Blalock 
et al. 1988). Hepatic metallothionein induction occurs after excessive dietary zinc intakes 
in rats, and is associated with hepatic zinc accumulation (McCormick et al. 1981). Other 
treatments that induce hepatic metallothionein, such as administration of interleukin- 1 , 
interleukin-6, and lipopolysaccharide, result in hepatic zinc accumulation and serum zinc 
depression (De et al. 1990, Huber and Cousins 1993). Taken together, these data suggest 
that metallothionein expression exerts a strong influence on tissue zinc distribution. 

Within cells, metallothionein also may function as a zinc reservoir from which 
this metal is made available for incorporation into apometalloenzymes or other 
metalloproteins. This idea is supported by the highly rapid exchange rates of zinc in 
metallothionein, which are far faster than the exchange rates from other proteins (Li et al. 
1980, Udom and Brady 1980). Further, incubation with Zn-metallothionein reconstitutes 
a number of enzymes and transcription factors (e.g., apocarbonic anhydrase and Spl) and 
rescues their activities (Li et al. 1980, Udom and Brady 1980, Zeng et al. 1991). 
Exchange rates are more rapid and more zinc is exchanged in the presence of oxidized 
glutathione, possibly linking zinc release to cellular redox status (Jiang et al. 1998). 
Interestingly, zinc is also liberated from metallothionein by a number of oxidants 
(Berendji et al. 1997, Fliss and Menard 1992). 

We undertook research concerning the role of metallothionein in regulation of 
zinc absorption due to the importance of zinc for health. Proper zinc nutrition is 
particularly important to support growth and immune functions (reviewed in Keen and 
Gershwin 1990, Rivera et al. 1995). In particular, zinc deficiency is known to retard 



growth and inhibit sexual maturity, both of which can be reversed to some degree with 
zinc supplementation (as reviewed in Prasad 1991). Further, supplemental zinc can help 
to alleviate secretory diarrhea and morbidity in third world countries (Sazawal et al. 
1995). Consequently, determining the mechanism(s) and regulation of zinc absorption 
provides information that can be used to efficiently and effectively maintain proper zinc 
homeostasis, and to support health. 

Metallothionein and Zinc in Defense Against Oxidative Stress 

The pathogenesis of aging, cancer, atherosclerosis, cataracts, neurodegenerative 
disorders, and ischemia-reperfusion injury are associated with oxidative stress (Blot et al. 
1993, Weidau-Pazos et al. 1996, Fraga et al. 1990, Rengstrom et al. 1992, Rosen et al. 
1993, Taylor et al. 1992). Oxidative stress occurs when the balance between oxidative 
attacks and oxidative defense systems favors oxidation. The mediators of oxidative 
damage include reactive oxygen species (i.e., H 2 2 , -0 2 and OH), and radical species that 
are not oxygen-centered (e.g., *CC1 3 ). These species can cause damage to lipids, proteins, 
and nucleic acids (Farber 1994, Loft et al. 1994, Oliver et al. 1990). Living organisms 
combat oxidative stress through the use of antioxidant nutrients such as tocopherols and 
ascorbate, as well as endogenous antioxidant scavengers like superoxide dismutase and 
glutathione (Yu 1994). When damage does occur, organisms have damage repair systems 
to fall back on. For example, DNA excision repair systems remove oxidized DNA bases, 
and glutathione peroxidase can convert lipid hydroperoxides formed by membrane 
oxidation to less reactive lipid hydroxides. Further, proteolytic and lipolytic enzymes 
degrade damaged macromolecules when damage is irreversible. But when these systems 
are overcome, the cell or organism may not survive. Since the generation of reactive 



8 



oxygen species is unavoidable, maximizing antioxidant defense systems has become a 
research priority (Cohen 1994). 

Adequate zinc nutrition may help protect against oxidative stress. Zinc-deficient 
rodents display markers of oxidative damage, and are more susceptible to subsequent 
oxidative stresses (DiSilvestro and Carlson 1993, Miceli et al. 1999, Oteiza and Keen 
1995). These results may reflect depressed activity of Cu/Zn superoxide dismutase, 
increased oxidation of sulfydryl groups that are normally protected by zinc binding, or 
increased exposure of zinc binding sites within proteins to copper- and iron-induced 
oxidation (as reviewed in Powell 2000). Increased oxidative stress also might be due to 
perturbation of a number of other zinc-dependent processes, including maintenance of 
cellular metallothionein (Blalock et al. 1988, Schroeder and Cousins 1990). This is due to 
reduced activation of metallothionein gene expression, as well as enhanced susceptibility 
of metallothionein protein to proteolysis when zinc is not available for binding (Smith et 
al. 1978, Feldman and Cousins 1976). Metallothionein expression may help defend 
against oxidative stress since metallothionein is capable of scavenging free radicals, and 
the hydroxyl radical in particular (Thornalley and Vasak 1985). 

Beyond preventing zinc deficiency, supplemental zinc provides additional 
protection against certain oxidative stresses (Blain et al. 1998, Dhawan and Goel 1995). 
Although the mechanism of protection is uncertain, it may include the induction of 
metallothionein protein. Metallothionein is induced by a number of chemicals that 
generate oxidative stress (Bauman et al. 1991, Satoh et al. 1996, Shiraishi et al. 1989; 
Tate et al. 1995). Preinduction of metallothionein by a number of agents (including zinc, 
other metals, hormones, and cytokines) is associated with protection against the toxicity 



of subsequent metal, chemical, and other stresses in cell culture and in vivo (e.g., Coppen 
et al. 1988, Moffat et al. 1996, Satoh et al. 1992, Schroeder and Cousins 1990). Similar 
results were seen when cells were transfected with metallothionein genes (Kaina et al. 
1990, Schwarz et al 1995). Other reports showed no protection by metallothionein 
expression against free radicals, however, casting doubt on metallothionein' s role in 
oxidative defense (Kaina et al. 1990, Kelley et al. 1988). 

Based on these observations, supplemental zinc and metallothionein expression 
may or may not affect the outcome of conditions characterized by oxidative stress 
(Oteiza et al. 1995, Prasad 1991). Characterization of zinc and metallothionein as 
participants in oxidative defense is important because oxidative damage is associated 
with so many disease processes. For example, oxidative stress is a component of 
inflammatory bowel disease, which is associated with a reduced metallothionein content 
of the bowel (Mulder et al. 1991). Also, free radical production and oxidative stress 
characterize many diseases of the liver, and the liver is an organ that contains high zinc 
and metallothionein concentrations after zinc supplementation (Cohen 1994). 

Hypotheses and Research Objectives 

Based on the information summarized above, we developed two hypotheses 
regarding the biochemical actions and interactions of zinc and metallothionein: 

1 . Zinc absorption is inversely related to intestinal metallothionein production, 
while tissue distribution of absorbed zinc is directly related to the 
metallothionein content of the tissue. 

2. Zinc and/or metallothionein protect mouse livers and hepatocyte cultures from 
oxidative damage. 



10 

We examined these hypotheses using relatively new models for biological 
research - transgenic mice. We used metallothionein transgenic overexpressing mice 
(designated TG), metallothionein knockout mice (i.e., null; designated KO), and their 
respective control strains in all of the studies that follow. Metallothionein transgenic mice 
carry 56 copies of the metallothionein- 1 gene in their genome, and those genes are 
responsive to the same stimuli that induce endogenous metallothionein- 1 genes (Palmiter 
et al. 1993). In contrast, metallothionein knockout mice produce no metallothionein- 1 or 
-2 protein under any conditions in the tissues we studied (Masters et al. 1994A). This 
allows us to assess the effects of a broad spectrum of metallothionein expression levels 
on zinc metabolism and oxidative stress. Further, studies with metallothionein null mice 
allow determination of biological effects of zinc that are independent of metallothionein. 



CHAPTER 2 

THE EFFECT OF METALLOTHIONEIN EXPRESSION ON ZINC ABSORPTION IN 

METALLOTHIONEIN TRANSGENIC AND METALLOTHIONEIN KNOCKOUT 

MICE 

Introduction 

The mechanisms that regulate zinc metabolism are not understood. When dietary 
zinc intake is restricted in experimental animals and humans, the efficiency of zinc 
absorption increases and endogenous zinc excretion decreases. Furthermore, zinc 
absorption is depressed after ingestion of zinc-rich diets. The biomolecules that mediate 
the regulation of zinc metabolism by the dietary zinc supply have not been fully 
described. The cytosolic protein metallothionein (MT) may be a principal participant in 
this regulation. This metalloprotein is inducible by many factors (stimuli) and may act as 
a zinc pool or buffer that is influenced by body zinc levels. In addition, the redistribution 
of endogenous zinc associated with stresses such as acute infection and physical trauma 
may require metallothionein. The induction is believed to involve interleukin-1, 
interleukin-6, and glucocorticoid hormone-mediated changes, all of which can be linked 
to elevated expression of metallothionein in the liver and other tissues (reviewed in 
Cousins 1989, 1996). 

Metallothionein transgenic mice (TG) and metallothionein knockout mice (KO) 
provide a unique model to study the effects of metallothionein expression on zinc 
absorption. Metallothionein transgenic mice have elevated metallothionein protein in 
many tissues, including the liver and intestine (Iszard et al. 1995, Liu and Klaassen 



11 






12 

1996). The larger cytosolic metallothionein pools might convey protection against zinc 
deficiency (Dalton et al. 1996). Conversely, metallothionein knockout mice allow 
examination of how zinc metabolism differs when no metallothionein is produced 
(Masters et al. 1994, Michalska and Choo 1993). Others have shown that KO mice have 
altered zinc metabolism, including inability to sequester zinc in the liver after injections 
of zinc or lipopolysaccharide (Coyle et al. 1995, Philcox et al. 1995). Further, 
hepatocytes from KO mice were incapable of accumulating zinc in response to 
interleukin-6 or dexamethasone treatment. Hence, significant perturbations of zinc 
metabolism occur in mice with altered metallothionein expression. This study was 
directed at determining the effects of altered metallothionein expression on zinc 
absorption. 

The intestine is a major control site for zinc homeostasis and is also a major 
metallothionein-expressing organ (Cousins 1989). While some data support an inverse 
relationship between intestinal metallothionein expression and zinc absorption (Hoadley 
et al. 1987, Hoadley et al. 1988, Menard et al. 1981, Smith and Cousins 1980), other data 
do not (Flanagan et al. 1983, Starcher et al. 1980). To examine how metallothionein 
influences the intestinal processing of zinc, a zinc dose was delivered by gavage (zinc 
tolerance test), and the increase in the serum zinc concentration was used as a measure of 
absorption. 

Materials and Methods 
Animals 

The founder mice used in this study were obtained from The Jackson Laboratory, 
Bar Harbor, ME. The transgenic mice (designated TG mice) were derived from the 



13 

C57BL/6 strain crossed with the SJL strain (Palmiter et al. 1993). The metallothionein 
knockout mice (designated KO mice) were derived from the 129/SvCPJ strain crossed 
with C57BL/6 (Masters et al. 1994). Mice of the appropriate background strains served 
as controls, designated CT and CK, respectively. Only adult male mice were used for 
experiments. They were housed in plastic cages with wood shavings as bedding and with 
a 12 h light/dark cycle. The mice had free access to a commercial diet (Laboratory 
Rodent Diet No. 5001, PMI Feeds, New Albany, IN). All experiments were started 
between 8:00 and 10:00 AM. Care and treatment of the mice received approval of the 
University of Florida Institutional Animal Care and Use Committee. 

Radioisotopes 

The a 32 P-dCTP was from Du Pont NEN (Boston, MA), and the 109 Cd (1.35 x 10 6 
kBq/nmol Cd) was from Isotope Product Laboratories (Burbank, CA). 

RNA Isolation and Northern Analysis 

Total RNA was isolated from intestine and liver using TRIzol reagent (Life 
Technologies, Gaithersburg, MD). Briefly, 50 to 100 mg of the proximal duodenum and 
the liver were homogenized in 2 mL of TRIzol reagent. After addition of chloroform, the 
RNA was processed and analyzed as described previously (Blanchard and Cousins 1996). 
Equal quantities of RNA from mice of each group were pooled and subjected to Northern 
analysis (20 ug total RNA per lane). Equal loading was confirmed by ethidium bromide 
staining. Northern blot analyses were carried out using a rat metallothionein- 1 cDNA 
probe (Blanchard and Cousins 1996). It was radiolabeled with a 32 P-dCTP using the RTS 
RadPrime DNA Labeling System (Life Technologies) as described previously 






14 

(Blanchard and Cousins 1996). The metallothionein cDNA probe hybridizes to 
metallothionein mRNA from the control mice and the KO mice. The metallothionein 
mRNA of the KO mice contains a premature stop codon, however, and can not be 
translated into MT protein. 

High Performance Liquid Chromatography of Intestinal Cytosol 

In some experiments, the mucosa was homogenized with a Potter Elvejhem tissue 
grinder using 2 volumes of ice-cold buffer (S-12 buffer, 154 mmol/L NaCl, 10 mmol/L 
TrisCl, 3 mmol/L NaN 3 , and 10 mmol/L MgS0 4 ) plus protease inhibitors (0.1 mmol/L 
phenylmethylsulfonyl fluoride, 1.2 umol/L leupeptin and 1.5 umol/L pepstatin A). After 
centrifugation at 100,000 x g (30 min), the cytosol fraction was filtered (0.22 urn) and 
200 uL was applied to two Superdex 75 chromatography columns (1 x 30 cm; Pharmacia 
Biotech, Piscataway, NJ) in series using an isocratic elution of S-12 buffer (Hempe and 
Cousins 1989). Metallothionein was measured in all fractions using the cadmium ( 109 Cd)- 
binding assay (Eaton and Toal 1982). 

Oral Zinc Dosing 

Mice that had been fasted overnight were administered 0.5 mmol Zn/kg body 
weight as ZnS0 4 in saline, or saline alone via a stomach tube and were killed 2 h later. 
Blood was obtained by cardiac puncture and serum was prepared for serum zinc analysis. 
Liver and intestinal zinc concentrations were measured to determine if the zinc dose 
produced a change in tissue uptake/retention. 



15 

Analytical Methods and Statistical Analysis 

109 Cd was measured using a Packard Cobra II gamma spectrometer equipped with 
a 3 inch crystal (Packard, Downers Grove, IL). Metallothionein protein was measured by 
the cadmium ( 109 Cd) binding assay (Eaton and Toal 1982). Briefly, tissue extracts are 
boiled, and the resulting supernatant is incubated with 109 Cd. After removal of unbound 
109 Cd using hemoglobin as a chelator, 109 Cd bound to MT is measured by y-counting, and 
converted to moles of MT using the Cd-MT binding stoichiometry of 7: 1. Total protein 
was measured by the method of Lowry et al. (1951). Serum zinc concentrations were 
measured by flame atomic absorption spectrophotometry (AAS) (Hempe and Cousins 
1989). Tissue zinc was also measured by AAS, after sections of liver and intestine were 
digested with acid (HN0 3 /H 2 S0 4 ; 3/1) as described previously (Dunn and Cousins 1989). 
Data were analyzed by ANOVA followed by the Student-Newman-Keuls multiple 
comparison test where appropriate (InStat, GraphPad, San Diego, CA). Logarithmic 
transformation of some data was used to obtain homogenous variances before analysis. 

Results 
Metallothionein Expression in Intestine and Liver after Oral Dosing 

Northern analysis of metallothionein mRNA in liver and intestine demonstrates 
both basal and zinc-induced levels of expression in these genotypes (Fig. 2-1). All groups 
expressed metallothionein mRNA, but TG mice had several-fold greater metallothionein 
expression than either of the control strains in both the intestine and liver.Metallothionein 
mRNA was also expressed in KO mice, but this message contains a premature stop 
codon and is not translatable. Zinc treatment resulted in induction of metallothionein 
mRNA in the intestine and liver of all mice, but expression was greatest by far in the TG 



16 

mice. These data confirm that KO mice, control mice (CK and CT), and TG mice 
represent groups with distinguishably different basal and zinc-induced metallothionein 
mRNA levels in both intestine and liver. Consequently, if metallothionein is important in 
regulating zinc absorption, these groups should display different absorption 
characteristics. 

The knockout mutation was confirmed by identifying metallothionein protein in 
size exclusion chromatography fractions of intestinal cytosol (Fig. 2-2). Metallothionein 
protein was measured in each 0.5 mL fraction using the 109 Cd binding assay (Eaton and 
Toal 1982). A large metallothionein peak is seen between 32 and 35 mL in the 
chromatography profile from TG mice, but none is identifiable in the profile from the KO 
mice. No peak was seen in profiles from zinc treated KO mice (data not shown). 
Therefore, although metallothionein mRNA is produced by KO mice, no metallothionein 



+ 



+ + + 




Zinc 



Intestine 




CK 



KO 



CT 



TG 



Liver 



Figure 2-1. Intestinal metallothionein mRNA in metallothionein knockout (KO), 
knockout control (CK), transgenic (TG), and transgenic control (CT) mice that 
consumed diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Equal 
amounts of total RNA were pooled from intestine of 3-5 mice per group and analyzed 
by northern analysis using a metallothionein- 1 cDNA probe, with P-Actin used for 
normalization. (A) KO and CK mouse intestine. Although metallothionein- 1 mRNA is 
present in KO mice, it is not translatable. (B) CT and TG mouse intestine. (C) 
Graphical representation of metallothionein- 1 mRNA expression as a function of 



17 



protein results from that message. Although metallothionein is the predominant 109 Cd 
binding compound in the cytosol, a small but finite amount of 109 Cd binding is seen in 
nearly all other fractions in the profiles of TG and KO mice (Fig. 2-2). Others have 
observed the same phenomenon (Liu et al. 1996A). Since no 109 Cd binding activity is 




18 20 22 24 26 28 30 32 34 36 38 40 
Elution Volume (ml_) 

Figure 2-2. Superdex 75 size exclusion chromatography of metallothionein (MT) in 
cytosol from intestine mucosa of metallothionein-transgenic (TG) and metallothionein- 
knockout (KO) mice 2 h after an oral dose of saline. Mucosal cytosol was separated by 
two superdex 75 columns run in series and 0.5 mL fractions were collected. The MT 
content of fractions 18 through 40 is expressed as fig MT equivalents. Metallothionein 
elutes between 32 and 35 mL. 



18 

seen in any chromatographic fractions in the KO mice, including the fractions that 
contain metallothionein in normal mice, any l09 Cd binding activity associated with KO 
mice cytosol should be considered background. These levels do not increase when zinc 
treatment is given (data not shown). To better define the metallothionein content of 
tissues we deducted the average 109 Cd binding value associated with the cytosol of KO 
mice from all groups when measuring metallothionein in the intestinal mucosa (0. 1 mg/g 
protein; 15 nmol/g protein) and liver (10 ug/g liver; 1.5 nmol/g liver). It is clear that the 
saline treated controls have little metallothionein protein present in intestinal mucosa 
(Fig. 2-3). TG mice, however, have significantly elevated metallothionein levels 
compared to CT mice. Zinc treatment significantly increased metallothionein in all but 



1.2 








c 1 ° 


] Saline 
1 Zinc 






CD 

o 0.8 

i_ 

Q. 


- 






o) 0.6 

I— 


- 






5 0.4 
E 0.2 


b 

* i 


a 


a 


0.0 


r— n ■ 


■ ~*~ ' 


T 



CK 



KO 



] Saline 
I Zinc 




Figure 2-3. Metallothionein content of intestine mucosa of metallothionein-transgenic 
and metallothionein-knockout mice after an oral zinc dose. Metallothionein (MT) was 
measured by the l09 Cd binding assay (mg MT/g mucosa protein) in the intestine mucosa 
of metallothionein-transgenic (TG), TG control (CT), metallothionein-knockout (CK), 
and KO control (CK) mice after an oral dose of either saline (□) or saline and 0.5 mmol 
Zn/kg bw (■). Data are reported as mean +/- SEM of 4-5 mice/group. A constant value 
of 0.1 mg MT/g mucosa protein was deducted from all measurements to account for the 
nonspecific 109 Cd binding observed in KO mouse mucosa. Data was analyzed by 
ANOVA followed by the Student-Newman-Keuls multiple comparison post test. Bars 
labeled with different letters within a graph are statistically different from each other (p 
< 0.05) 



19 

the KO mice group. Again, the induction was greatest in TG mice, being eight fold 
higher than the zinc treated CT mice. Overall metallothionein protein was not present in 
KO mice, was only present in CT and CK mice after zinc treatment, and was always 
greatest in TG mice. 

Metallothionein was also measured in liver (Fig. 2-4). Unlike in the intestine, 
liver metallothionein in saline treated controls was greater than in KO mice, and similar 
to TG mice. Zinc treatment elevated the mean value of metallothionein 5-fold in CT (p > 
0.05) and 10-fold in TG mice livers, but the increase was only significant in TG mice. No 
increase in liver metallothionein was seen in CK mice after zinc treatment. This was not 
completely unexpected, however, since differences in zinc induction of liver 



180 
150 

u- 

~ 1 
CO 

■i 90" 
60 
30 




CO 

c 



] Saline 
I Zinc 



b 




CKO 



KO 



CTG 



TG 



Figure 2-4. Metallothionein content of liver in metallothionein-transgenic and 
metallothionein-knockout mice after an oral zinc dose. Metallothionein (MT) was 
measured by the i09 Cd binding assay (ug MT/g liver) in the liver of metallothionein- 
transgenic (TG), TG control (CT), metallothionein-knockout (KO), and KO control 
(CK) mice 2 h after an oral dose of either saline (□) or saline and 0.5 mmol Zn/kg bw 
(■). Data are reported as mean +/- SEM of 4-5 mice/group. A value of 10 ug MT/g 
liver was deducted from all measurements to account for the nonspecific 109 Cd binding 
observed in KO mouse liver. TG and CT data was log l0 transformed prior to statistical 
analysis to achieve homogeneous variances. Data was analyzed by ANOVA followed 
by the Student-Newman-Keuls multiple comparison post test. Bars labeled with 
different letters within a graph are statistically different from each other (p < 0.05). 



20 

metallothionein have been seen among mouse strains (Fair and Hunt 1989). Hence, each 
group had a different amount of metallothionein protein present in intestine and liver 
after zinc treatment. Since metallothionein may provide a zinc storage pool in these 
organs, mice with the greatest metallothionein production (TG mice) have a greater 
capacity to deal with a zinc load than those with the least metallothionein (KO mice). 

Intestine, Liver and Serum Zinc Responses Two Hours after the Oral Zinc Dose 

Although metallothionein in intestine and liver differed among groups, these 
differences were not correlated to detectable differences in intestine and liver zinc 
concentrations (data not shown). When saline was given, all mice had roughly equivalent 
zinc concentrations in both intestine and liver (approximately 0.48-0.62 umol Zn/g and 
0.37-0.59 umol Zn/g, respectively). No significant elevation of liver zinc was seen after 
zinc treatment. All groups had significantly elevated zinc concentrations in intestine after 
zinc treatment, but there were no significant differences among the zinc-treated groups 
except in the KO group. In the KO mice, zinc treatment increased the intestinal zinc 
concentration significantly compared to the zinc-treated CK mice (1.37 ± 0.22 vs. 0.86 ± 
0. 1 1 umol Zn/g, respectively; p < 0.05). In contrast, the intestinal zinc concentrations in 
zinc-treated CT and TG mice were similar (0.97 ±0.18 and 1.03 ± 0.18 umol Zn/g, 
respectively). Hence, absence of metallothionein resulted in a detectable increase in zinc 
accumulation in intestine. However, overexpression in the TG mice did not influence 
intestinal zinc retention. This suggests that metallothionein does not alter zinc 
metabolism simply by sequestering zinc in the intestine. The change in serum zinc 
concentration two hours after the oral zinc dose was used as an indicator of the quantitiy 
of zinc absorbed (Fig. 2-5). In contrast to tissue zinc, serum zinc was markedly affected 



21 

by metallothionein expression. Although all groups had similar serum zinc 
concentrations when given saline (15-30 umol/L), mice with greater metallothionein 
expression had lower concentrations after zinc treatment than mice with less 
metallothionein expression. Zinc treated control strains had serum zinc concentrations 4 
to 5 times higher than saline treated controls. KO mice, however, had 10-fold greater 
serum zinc values after zinc treatment. Conversely, TG mice had only 2.3-fold greater 
serum zinc concentrations after zinc treatment. Thus, serum zinc concentrations were 
inversely proportional to the amount of intestinal metallothionein expressed. This 
relationship is illustrated in Fig. 2-6. 

Discussion 
Our hypothesis was that intestinal metallothionein acts as a negative regulator of 
zinc absorption. This relationship has been examined in the past, but with conflicting 




Figure 2-5. Serum zinc in metallothionein-transgenic and metallothionein-knockout 
mice after an oral zinc dose. Zinc was measured by atomic absorption 
spectrophotometry (umol zinc/L) in serum of metallothionein-transgenic (TG), TG 
control (CT), metallothionein-knockout (KO), and KO control (CK) mice 2 h after and 
oral dose of either saline (□) or saline and 0.5 mmol Zn/kg bw (■). Data are reported 
as mean +/- SEM of 4 mice/group. Data were analyzed by ANOVA followed by the 
Student-Newman-Keuls multiple comparison post test. Bars labeled with different 
letters within a graph are statistically different from each other (p < 0.05). 



22 

results. For instance, Dr. Cousins' laboratory (Hoadley et al. 1987, Menard et al. 1981, 
Smith and Cousins 1980, Smith et al. 1978) found that the quantity of zinc absorbed by 
the isolated perfused rat intestine was inversely related to the zinc content of the diet 
consumed prior to the experiments. In addition, giving rats a large zinc dose (i.p.) 18 h 
prior to experiments depressed zinc absorption. Since zinc absorption was inversely 
proportional to intestinal metallothionein throughout those experiments, it was proposed 
that metallothionein serves as a damper of zinc absorption. Similarly, studies where rats 
consumed diets ranging from 5 to 80 mg Zn/kg also showed that zinc absorption was 
inversely related to metallothionein-bound zinc (Coppen and Davies 1987). Furthermore, 
Hoadley et al. (1988) found that elevated metallothionein levels in intestines of fasted 
rats were associated with greater mucosa to lumen transfer of absorbed zinc by the 
isolated perfused rat intestine. They proposed that metallothionein depresses zinc 
absorption by providing a sink that holds zinc in the intestine, allowing more opportunity 
for transfer of zinc back into the lumen. 

Contrary to the results cited above, other studies found no relationship or positive 
correlation between intestinal metallothionein and zinc absorption. For example, bacterial 
infection, endotoxemia, and interleukin- 1 administration to rats all elevated liver 
metallothionein, and resulted in 50-100% greater zinc absorption and liver zinc 
accumulation from 65 Zn doses (Kincaid et al. 1976, Pekarek and Evans 1975, 1976). 
Intestinal metallothionein expression was not evaluated in those experiments, but 
endotoxin has been shown to induce both metallothionein- 1 and metallothionein-2 in 
mouse intestine (De et al. 1990). Also, interleukin- 1 is thought to be a mediator of 
metallothionein induction by LPS in some tissues, and thus may induce the protein in the 



23 

intestine as well (reviewed in Cousins 1996). Furthermore, small zinc doses (0.2 umol/kg 
body weight; i.p.) caused metallothionein induction in the mouse intestine and 
corresponded to enhanced zinc absorption 18 h later (Starcher et al. 1980). Flanagan and 
coworkers (1983) observed no difference in zinc uptake or absorption in relation to 
intestinal metallothionein in intestinal perfusion experiments with mice. They did, 
however, see greater zinc absorption in zinc-deficient vs. control mice when doses of 



X KO 

• CK 
A CT 

♦ TG 




Intestine Metallothionein (mg/g protein) 

Figure 2-6. Serum zinc concentration as a function of intestine metallothionein content 
in zinc-treated mice. Response of serum zinc (Y) vs. intestinal metallothionein content 
(X) in metallothionein-transgenic (TG), TG control (CT), metallothionein-knockout 
(KO), and KO control (CK) mice 2 h after gavage with 0.5 mmol Zn/kg bw. 
Metallothionein (MT) is expressed as mg MT/g mucosa protein and serum zinc as 
umol Zn/L. The inverse relationship between intestinal MT and serum zinc can be 
expressed mathematically with good fit (Y = 1690/X + 0.75; r 2 = 0.94). 



24 

zinc were delivered by gavage. They also demonstrated that differences exist in zinc 
absorption characteristics between rats and mice, particularly the response of increased 
absorption during zinc deficiency. 

Although the studies cited above focused on the effect of metallothionein on zinc 
absorption, the methods used to alter intestinal metallothionein levels varied. Treatments 
used to induce the protein included intraperitoneal, intragastric, and dietary doses of zinc, 
fasting, bacterial infection, lipopolysaccharide and interleukin-1 administration, and 
various forms of physical stress. Although these treatments manipulate metallothionein 
expression, each has effects not related to this protein that may cause physiological 
changes and complicate interpretation of the results. Using knockout and transgenic 
mouse models, it is possible to focus on zinc absorption as directly related to 
metallothionein expression. 

Giving animals a large oral dose of zinc by gavage, we were able to determine the 
effects of metallothionein induction on zinc absorption by measuring serum and tissue 
zinc concentrations. This avoids the potential for isotope dilution, which can cloud 
interpretation of radioisotopic tracer studies using 65 Zn. We have used the oral dosing 
approach previously (Menard et al. 1981). It is equivalent to the zinc tolerance test used 
with humans (Sullivan et al. 1979). We used fasting and dosing in saline to prevent 
nonspecific binding of zinc to food in the gut, and to allow for a maximal gastric 
emptying rate. The 2 h time point used was determined to be the time point of maximal 
serum zinc response in these mouse strains (data not shown), and agrees with data from 
rats (Menard et al. 1981) and humans (Sullivan et al. 1979, Valberg et al. 1985). Further, 
all dosing was done between 8 AM and 10 AM. The 0.5 mmol/kg dose given is 2.5 to 3.1 



25 

times greater than the typical dietary zinc intake of these mice (0.17 to 0.22 mmol/kg 
body weight). Although greater than the typical intake, this dose is attainable through the 
diet, and is therefore nutritionally relevant. Further, since the dose produced fivefold to 
tenfold increases in serum zinc, we anticipate that smaller doses will also result in 
significant differences, albeit smaller. Menard et al. (1981) showed that intestinal 
metallothionein synthesis in rats was increased by 3 h after an oral zinc dose is given. 
Furthermore, a second dose of zinc caused induction of metallothionein synthesis and 
resulted in better regulation of serum zinc concentrations. In the present experiments, 
serum zinc doubled in TG mice and increased 10-fold in KO mice when zinc was 
delivered by gavage. We interpret the inability of the KO mice to handle the zinc load 
compared to the TG mice to the difference in metallothionein expression. Specifically, 
the TG mice controlled serum zinc concentrations more tightly than did the KO mice. 
Serum zinc concentrations remain elevated for a considerable time after the oral dose. 
We cannot rule out that the observed differences wee related to different kinetics of 
absorption in these genotypes, however. 

A drawback of this approach is that the role of other tissues in clearance of zinc 
from the circulation cannot be explained adequately. Since the gene addition in TG mice 
and gene deletion in KO mice are not tissue specific, we cannot rule out the possibility 
that MT expression in some other tissue affected zinc clearance from the serum. 
However, we did measure the zinc content of the liver, the main zinc storage organ and 
the key organ in the regulation of zinc metabolism (Cousins 1996, Coyle et al. 1995). In 
this study, no change in liver zinc was detected between saline or zinc-treated mice, and 
no difference was seen among groups of zinc-treated mice. This is in agreement with data 



26 

collected in rats, where hepatic accumulation of gavaged zinc was not observed until nine 
hours after dosing (McCormick et al. 1981). Since the liver can act rapidly to regulate 
zinc metabolism, yet did not show an elevation in zinc concentration, it is unlikely that an 
organ with less influence on zinc metabolism caused the differences seen in serum zinc. 

In the time that has elapsed since the publication of the results found above 
(Davis et al. 1998), several reports have been forwarded regarding the role of 
metallothionein in zinc absorption. A number of these studies used a separate strain of 
metallothionein knockout mice (Michalska and Choo 1993). Our findings regarding the 
serum zinc response to the oral zinc dose was confirmed in these studies, i.e., knockout 
mice had greater serum zinc levels than control mice over a range of oral doses that 
began two orders of magnitude lower than in our studies (Coyle et al. 1999). Further, 
their studies confirmed greater zinc accumulation in duodenum and jejunum of KO mice 
than CK mice. These researchers also found lower accumulation of a radiolabelled dose 
in tissues other than the intestine (liver, skin, muscle, kidneys and pancreas) at 4 h post 
dose, which they interpreted as reduced absorption. However, KO mice lack the ability to 
sequester zinc in several organs (as determined by the researchers mentioned above, as 
well as by us in chapters 3, 4, and 5 of this dissertation), including the liver, which 
renders measurement of tissue zinc accumulation unsuitable as an index of intestinal zinc 
absorption in this model. 

Within intestinal cells, higher intakes of zinc may be processed via a mechanism 
that involves metallothionein. Since elevated intestinal metallothionein levels were not 
associated with greater intestinal zinc accumulation, metallothionein does not seem to act 
simply as a zinc sequestrant. Metallothionein may act as a zinc pool from which zinc is 



27 

highly available for transport back to the lumen, as suggested by Hoadley et al. (1988). 
Without metallothionein, the KO mice may be unable to maintain a satisfactory 
mucosa-to-lumen zinc flux. This might explain why KO mice have elevated zinc levels in 
serum and intestine. Since metallothionein mRNA levels are also induced by zinc in 
humans (Sullivan and Cousins 1997), we expect that metallothionein would affect zinc 
absorption by the human intestine as well. 



CHAPTER 3 

REGULATION OF METALLOTHIONEIN EXPRESSION AND ZINC METABOLISM 

BY DIETARY ZINC IN METALLOTHIONEIN TRANSGENIC AND 

METALLOTHIONErN KNOCKOUT MICE 

Introduction 

Homeostatic regulation of zinc metabolism by the dietary zinc supply is believed 
to involve the protein metallothionein (as reviewed in Davis and Cousins 2000). 
Metallothioneins are cysteine-rich, low molecular weight (6-7 kDa) metal binding 
proteins that can bind up to seven atoms of zinc per molecule of protein (as reviewed in 
Dunn et al. 1987). Metallothioneins are thought to provide a cellular zinc binding pool 
that may be influenced by body zinc levels (as reviewed in Davis and Cousins 2000). 
Redistribution of endogenous zinc is associated with stresses such as acute infection and 
physical trauma. Redistribution is believed to involve IL-1, IL-6, and glucocorticoid 
hormone mediated changes in zinc metabolism, all of which are linked to elevated 
expression of metallothionein in the liver and other tissues (reviewed in Cousins 1989 
and 1996). Metallothionein may also be involved in regulation of dietary zinc absorption 
in the intestine, and affect accumulation of dietary zinc in the liver (reviewed in Davis 
and Cousins 2000). Specifically, induction of intestinal metallothionein may reduce the 
efficiency of zinc absorption during times of elevated zinc intake. 

Previous studies firmly established the association of metallothionein induction 
with cellular zinc accumulation and bodily zinc redistribution (reviewed in Davis and 
Cousins 2000). Recent studies using metallothionein overexpressing transgenic mice (TG 



28 



29 

mice) and metallothioein knockout mice (KO mice) have more directly analyzed the role 
of metallothionein in zinc metabolism (Masters et al. 1994, Palmiter et al. 1993). Results 
from these studies confirmed the role of metallothionein expression in protection against 
severe zinc deficiency (Andrews and Geiser 1999, Dalton et al. 1996, Kelly et al. 1996). 
Other studies confirmed the necessity of metallothionein expression for zinc 
redistribution in response to immune stress, oxidative stresses and fasting (Davis et al. 
submitted, Philcox et al. 2000, Rofe et al. 1996). Metallothionein 's relationship to 
suppression of zinc absorption is still under debate (Davis et al. 1998, Coyle et al. 1999). 
Questions remain regarding metallothionein's role in tissue distribution of dietary 
zinc. Previous studies from this lab found that metallothionein induction after acute 
elevations in zinc intake was associated with accumulation of zinc in rat liver and 
intestine. Whether metallothionein induction is responsible for this increase in hepatic 
zinc, or whether it is only responding to the elevated hepatic zinc in not clear. Further, 
the effect of metallothionein expression on zinc distribution in mice during chronic 
exposure to elevated dietary zinc intakes is unknown. In these experiments we studied the 
interrelationship of metallothionein expression and chronic exposure to a spectrum of 
dietary zinc intake levels in metallothionein transgenic and metallothionein knockout 
mice in order to monitor this mode of regulation more closely. The results suggest that 
metallothionein expression affects tissue zinc accumulation only at highly elevated zinc 
intakes. Metallothionein appeared to regulate its own expression, however. This might 
have occurred through greater accumulation of cytosolic zinc. If so, metallothionein 
expression may alter the expression of other zinc-regulated genes. 



30 

Materials and Methods 
Animals 

Metallothionein knockout and metallothionein transgenic mice used in this study 
were derived from founder mice purchased from The Jackson Laboratory (Bar Harbor, 
ME). The metallothionein overexpressing mice (designated TG mice) were generated in 
C57BL/6 mice crossed with SJL mice (Palmiter et al. 1993), whereas metallothionein 
knockout mice (designated KO) were generated in 129/SvCPJ mice (Masters et al. 1994). 
C57BL/6 mice (designated CT) and 129S3/SvCPJ mice (designated CK) served as 
controls, respectively. Only 7-9 week old male mice were used in these experiments. 

Experimental Design 

Mice were housed singly in stainless steel hanging cages with a 12 h lightdark 
cycle. During experiments mice were given access to deionized water and semipurified 
diet based on the AIN-76A formulation (AIN 77; Research Diets, New Brunswick, NJ) 
with one of five zinc contents: 10, 50, 100, 200 and 300 mg zinc/kg diet (designated Zn 10 , 
Zn 50 , Zn 100 , Zn 200 and Zn 300 , respectively). Initially, mice were given free access to the 
Zn 10 diet and deionized water for seven days. For seven days thereafter mice were given 
free access to the Zn 10 , Zn 50 , Zn l00 , Zn 200 , or Zn 300 diet. All mice were killed between 8 
AM and noon following completion of the dietary treatment. Care and treatment of the 
mice received approval of the University of Florida Institutional Care and Use 
Committee. 
RNA Isolation and Northern Analysis 

Total RNA was isolated from intestine and liver using TRIzol reagent (Life 
Technologies, Gaithersburg, MD). Briefly, 50-100 mg of the proximal duodenum and 



31 

the liver were homogenized in 2 mL of TRIzol reagent. After addition of chloroform, the 
RNA was processed and analyzed as described previously (Davis et al. 1998). Equal 
quantities of RNA from mice of each group were pooled and subjected to northern 
analysis (20 ug total RNA per lane). Equal loading was confirmed by ethidium bromide 
staining. Northern blot analyses were carried out using a rat metallothionein-1 cDNA 
probe (Blanchard and Cousins 1996). This was radiolabeled with a 32 P-dCTP (Du Pont 
NEN, Boston, MA) using the RTS RadPrime DNA Labeling System (Life Technologies) 
as described previously (Blanchard and Cousins 1996). The metallothionein cDNA probe 
hybridizes to both the normal metallothionein mRNA of the control and TG mice, and 
the disrupted MT mRNA of the KO mice. Northern blots were also hybridized with a (3- 
actin probe, and the P-actin signal was used for normalization. Densitometry of the 
autoradiographs was performed by scanning the film and measuring the relative intensity 
using Intelligent Quantifier software (Bio Image, Ann Arbor, MI). 

Analytical Methods and Statistical Analysis 

Metallothionein was measured as described previously (Davis et al. 1998) using 
the cadmium ( 109 Cd) binding assay (Eaton and Toal 1982). Total protein was measured 
by the method of Lowry ( 1 95 1 ). Serum zinc concentrations were measured by flame 
atomic absorption spectrophotometry (AAS) (Hempe and Cousins 1989) after dilution 
with deionized water. Tissue zinc was measured by AAS after sections of liver and 
intestine were digested with HN0 3 /H 2 S0 4 (3/1) as described previously (Dunn and 
Cousins 1989). Data were analyzed by two way ANOVA (2x5) with SigmaStat software 
(Jandel Scientific, San Rafael, CA) to determine specific main effects and interactions 
using genotype and dietary zinc as independent variables using. The Tukey-Kramer 



32 



multiple comparison test was used to determine significant differences between specific 
groups (p < 0.05). 



mg Zn/kg diet 
MT- 1 mRNA 

(3-Actin mRNA 

B 

mg Zn/kg diet 
MT-1 mRNA 

(3-Actin mRNA 



CK 

10 50 100 200 300 



KO 

50 100 200 300 




CT 

10 50 100 200 300 



TG 

10 50 100 200 300 





10 50 100 200 300 
mg zinc/kg diet 



Figure 3-1. Intestinal metallothionein mRNA in metallothionein knockout (KO), 
knockout control (CK), transgenic (TG), and transgenic control (CT) mice that consumed 
diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Equal amounts of total 
RNA were pooled from intestine of 3-5 mice per group and analyzed by northern analysis 
using a metallothionein- 1 cDNA probe, with (3-Actin used for normalization. (A) KO and 
CK mouse intestine. Although metallothionein- 1 mRNA is present in KO mice, it is not 
translatable and therefore does not give rise to metallothionein protein. (B) CT and TG 
mouse intestine. (C) Graphical representation of metallothionein- 1 mRNA expression as 
a function of dietary zinc content. 






33 

Results 

Intestinal metallothionein mRNA was visibly upregulated by dietary zinc in CK 
and CT mice fed the Zn 100 through Zn 300 diets (Fig. 3-lA,B). Dietary zinc induced 
metallothionein mRNA more strongly and at lower zinc intakes in TG mice, but had little 
effect on metallothionein mRNA in KO mice. The proportional increases in 
metallothionein mRNA expression with dietary zinc intake were greater in mice with 
greater metallothionein expression (TG > CT and CK > KO) (Fig. 3-1C). The 
metallothionein mRNA expression pattern was similar in liver of KO, CK and CT mice 
(Fig. 3-2A,B). Metallothionein mRNA expression in TG liver was very high at all dietary 
zinc intakes, but relatively constant until the Zn 300 diet was consumed. 

Intestinal metallothionein protein was measured in all genotypes (Fig. 3-3A,B). 
The minimal values for metallothionein protein in KO mice (Fig. 3 -3 A) were shown to 
be assay background only (Davis et al. 1998). Considering this, little metallothionein 
expression was detected in CT or CK mice consuming the Zn 10 , Zn 50 or Zni 00 diets (Fig. 
3-3 A,B). Expression of metallothionein protein was directly regulated by dietary zinc in 
mice consuming the Zn 200 and Zn 300 diets. Similar to the response of metallothionein 
mRNA, intestinal metallothionein protein was induced at lower dietary zinc intakes and 
to a greater extent in TG mice. 

Intestine zinc concentrations were significantly affected by dietary zinc (p = 
0.00008) in CT and TG mice. Intestine zinc contents appear to be associated with 
intestinal metallothionein protein levels (Fig. 3-3A-D). Zinc content started to increase in 
all mouse intestines at the same dietary zinc level that metallothionein induction 



34 



occurred. The increase in intestine zinc was greater in TG mice (except with the Zn 300 
diet), and no increase was seen in KO mice. 

In contrast to the above results, the effects of dietary zinc on metallothionein 
expression and zinc content in liver differed between the two control species (Fig. 3-4A- 
D). Dietary zinc did not affect liver metallothionein levels in CK or KO livers (Fig. 3- 
4A). Also, metallothionein levels in CK mice did not exceed the assay background seen 
in KO mice. In contrast, TG mice had greater liver metallothionein values than CT mice 
at all zinc intakes, but zinc did not induce metallothionein above basal levels until diets 
containing 300 mg Zn/kg were consumed (Fig. 3-4B). 

Liver zinc was not altered by dietary zinc or metallothionein expression except at 
the highest dietary zinc intake (Fig. 3-4C,D). Liver zinc was not affected by dietary zinc 
or genotype in CK and KO mice (Fig. 3-4C). Only the high dietary zinc concentration 
(Zn 300 ) elevated liver zinc, and only in TG mice (Fig. 3-4D). 



A 

mg Zn/kg diet 

MT-1 mRNA 

P-Actin mRNA 

B 

mg Zn/kg diet 
MT- 1 mRNA 

(3-Actin mRNA 



CK KO 

10 50 100 200 300 10 50 100 200 300 



CT TG 

10 50 100 200 300 10 50 100 200 300 




« liM| 



Figure 3-2. Liver metallothionein mRNA in metallothionein knockout (KO), knockout 
control (CK), transgenic (TG), and transgenic control (CT) mice that consumed diets 
containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. mRNA was analyzed by 
northern analysis as described in Fig. 3-1. (A) KO and CK mouse intestine. (B) CT 
and TG mouse liver. 



35 



Serum zinc was affected by dietary zinc in all genotypes (Fig. 3-5 A,B). Serum 
zinc began to rise in CT and CK mice consuming Zn 100 and Zn 200 diets, respectively, and 



1000 



a 800 

1 

P. 600 

H 

S 400 



H 200 



I CK 

D ko 




a a 
10 



' Dct B 

■ TG 

a,b 
a 

a a aj ftj 


b 




c 
1 



300 



10 50 100 200 300 



3000 
2500 

M 2000 

S 

2 1500 

3 1000 

500 



I CK 

□ KO 



tad 





10 50 100 200 300 10 50 

mg zinc/kg diet 



100 200 300 



Figure 3-3. Intestinal mucosal metallothionein protein and zinc in metallothionein 
knockout (KO), knockout control (CK), transgenic (TG), and transgenic control (CT) 
mice that consumed diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. 
Mucosal metallothionein in (A) CK and KO mouse intestine or (B) CT and TG mouse 
intestine. Values found for KO mice reflect assay background only. Intestinal zinc in 
(C) CK and KO mouse intestine or (D) CT and TG mouse intestine. Data are 
presented as means ± SE of 3-6 mice/group. Statistical differences (p < 0.05) were 
determined by two-way ANOVA followed by the Tukey-Kramer post test 



36 

continued to rise with increasing dietary zinc level. The effect of diet alone was very 
significant (p = 0.0000005). No differences were seen between CK and KO mice. Serum 
zinc of TG mice was slightly elevated comparedto CT mice consuming Zn 10 -Zn 200 diets. 
When Zn 300 diets were consumed, however, serum zinc was greater in CT mice than TG 
mice. 

Discussion 
Metallothionein has long been implicated as a key biomolecule in regulation of 
zinc homeostasis. Specifically, many lines of evidence point to metallothionein as a 
component of the machinery involved in intracellular zinc accumulation (as reviewed in 
Davis and Cousins, 2000). Here, we confirm that KO mice are unable to accumulate zinc 
in the intestine in response to chronic exposure to elevated dietary zinc, as was found 
previously in a different metallothionein KO mouse model (Tran et al. 1998). This report 
extends this relationship to TG mice, which generally accumulated more intestinal zinc 
than controls. These data show that zinc accumulation is affected by metallothionein 
expression over a wide range of intestinal metallothionein contents. Further, our data 
from TG mice show for the first time that the level of hepatic metallothionein expression 
dictates the livers ability to accumulate zinc during chronic exposure to elevated dietary 
zinc. This expands on the relationship previously seen after zinc injections in control and 
KO mice, where KO mice were unable to accumulate zinc in the liver (Rofe et al. 1996). 
If hepatic uptake of zinc is important during infection and trauma, metallothionein might 
be crucial for processes that depend on this influx of zinc. Such a role has been suggested 
for metallothionein expression in rat liver during regeneration and the acute phase 
response (Arora et al. 1998, Dunn and Cousins 1989, Ohtake et al. 1978). A similar role 



37 



would be predicted in other metallothionein-expressing organs. As such, overexpression 
of metallothionein may be beneficial to liver regeneration, or conditions associated with 
the acute phase response. 

We also discovered that induction of liver and intestine metallothionein mRNA 
and protein occurred at lower dietary zinc intakes in animals with greater metallothionein 

200 





10 50 100 200 300 



10 50 100 200 300 



DC 



2000 
1800 
1600 
1400 



g 1200 

jj 1000- 
o 

E 
c 



800 
600 
400 
200 





■ CK 
□ KO 



miiil 




10 50 100 200 300 10 50 

mg zinc/kg diet 



100 



200 300 



Figure 3-4. Liver metallothionein protein and zinc in metallothionein knockout (KO), 
knockout control (CK), transgenic (TG), and transgenic control (CT) mice that consumed 
diets containing either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Metallothionein in (A) 
CK and KO mouse liver or (B) CT and TG mouse liver. Values found for KO mice 
reflect assay background only. Zinc concentration in (C) CK and KO mouse liver or (D) 
CT and TG mouse liver. Data are presented as means ± SE of 3-6 mice/group. Statistical 
differences (p < 0.05) were determined by two-way ANOVA followed by the Tukey- 
Kramer post test. 






38 

production (TG < CT and CK < KO; Fig. 3-1 & Fig. 3-2). Similar results were seen for 
metallothionein protein (Fig. 3-3A,B & 3-4A,B). These data suggest that metallothionein 
enhances its own expression, possibly by facilitating cellular zinc accumulation. In this 
hypothesis, accumulation of cellular zinc in the metallothionein-zinc pool provides a 
labile zinc pool that may interact with other pools (as illustrated in Davis and Cousins 
2000). One such pool is the nucleus, where dietary zinc rapidly accumulates in a number 
of organs (Cousins and Lee- Ambrose 1992). One of the biomolecules that the 
metallothionein-Zn pool may interact with is the zinc-finger transcription factor MTF-1. 
MTF- 1 contains a zinc-binding site that may be sensitive to cellular zinc levels (as 
reviewed in Andrews 2000). Zinc from the metallothionein-Zn pool may activate MTF-1, 
resulting in subsequent transcriptional activation of the metallothionein gene through 
numerous metal response elements (MREs) in the metallothionein gene promoter. This 




10 50 100 200 300 10 50 100 200 300 

mg zinc/kg diet 

Figure 3-5. Serum zinc in metallothionein knockout (KO), knockout control (CK), 
transgenic (TG), and transgenic control (CT) mice that consumed diets containing 
either 10, 50, 100, 200 or 300 mg Zn/kg for 7 d. Serum zinc in (A) CK and KO mice 
or (B) CT and TG mice. Data are presented as means ± SE of 4-6 mice/group. 
Statistical differences (p < 0.05) were determined by two-way ANOVA followed by 
the Tukey-Kramer post test. 



39 

process would enhance production of metallothionein protein, which, after further zinc 
binding, may fuel even greater metallothionein gene transcription. Cycling through this 
pathway would amplify metallothionein levels, and as we observed, more amplification 
would occur in mice with greater metallothionein expression. 

The fact that metallothionein protein expression was elevated only with chronic 
dietary zinc supplementation is in line with previous mouse experiments (Olafson 1983, 
Tran et al. 1998). Tissue zinc levels were only elevated at high zinc intakes, also. Further, 
KO mice maintained control over serum zinc, liver zinc and intestine zinc levels as well 
as CK mice did. These results suggest that mice can maintain tight control of zinc 
homeostasis over a large range of zinc intakes without metallothionein induction. Many 
zinc transporter molecules have been identified recently, and it is likely that regulation of 
the activity or expression of those proteins mediated zinc homeostasis over that range of 
zinc intakes (reviewed in McMahon and Cousins 1998, Cousins and McMahon 2000). 
The high correlation of zinc transporter 1 expression (Davis et al. 1998) and zinc 
transporter-2 expression (Liuzzi et al. 2000) with metallothionein expression supports the 
involvement of zinc transporters and metallothionein expression in zinc homeostasis. 
Metallothionein appears to become an important mediator of cellular zinc homeostasis at 
very high dietary zinc intakes, however (Davis et al. 1998). This is not to say that 
metallothionein plays no role in zinc homeostasis at lower dietary zinc levels. It has been 
shown that metallothionein expression provides partial protection against zinc deficiency 
(Andrews and Geiser 1999, Dalton et al 1996, Kelly et al. 1996). It also is necessary for 
redistribution of zinc during periods of stress (Cousins and Leinart 1988, Davis et al. 
submitted, Dunn and Cousins 1989, Philcox et al. 1995). When considering the typical 



40 

range of dietary zinc intakes and in the absence of metallothionein-inducing stresses, 
however, the effect of metallothionein expression on zinc homeostasis is minimal. 

In conclusion, we found that metallothionein expression alters tissue zinc 
accumulation, but only at highly supplemental zinc intakes. At lower intakes 
metallothionein was not a factor. We found evidence that metallothionein expression 
might act in a positive feedback loop to regulate its own expression, and that this occurrs 
to a greater degree in mice with a greater number of MT gene copies. If this hypothesis is 
true, metallothionein may also be involved in regulation of other zinc responsive genes. 






CHAPTER 4 
THE EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND 

SUPPLEMENTAL DIETARY ZINC IN PROTECTION AGAINST 

HEPATOTOXICITY IN METALLOTHIONEIN TRANSGENIC AND 

METALLOTHIONEIN KNOCKOUT MICE 

Introduction 

Zinc and metallothionein are implicated in cellular defense against a number of 
cytotoxic agents. There is evidence that supplemental zinc and overexpression of 
metallothionein help protect cells and organisms from a number of stresses. For example, 
administration of pharmacological zinc doses protects rodents from the toxicity of certain 
metals and other chemicals, some of which cause oxidative stress (Blain et al. 1998, 
Chvapil et al. 1973, Dhawan and Goel 1995, Powell et al. 1994). Similar results were 
seen in cell culture (Coppen et al. 1988, Liu et al. 1991, Tate et al. 1999). The 
mechanism(s) through which zinc provides protection is uncertain. Zinc may protect 
sulfhydryl groups from oxidation, may limit the redox reactive metal content of tissues, 
or may elevate the activity of antioxidant enzymes (Coppen et al. 1988, Davis, C. D. et 
al. 2000, Olin et al. 1995). Many believe that supplemental zinc provides antioxidant 
protection through its powerful induction of metallothionein gene expression. 

Preinduction of metallothionein by a number of metals (including zinc), 
hormones, cytokines and other chemicals is associated with protection from the toxicity 
of subsequent metal, chemical, and other stresses in cell culture and in vivo (Blain et al. 
1998, Coppen et al. 1988, Kelley et al. 1988, Liu et al. 1991, Mello-Filho et al. 1988, 
Moffat et al. 1996, Naganuma et al. 1985, Satoh et al. 1988, Schroeder and Cousins 



41 



42 

1990). Several experiments in cell cultures transfected with metallothionein genes (Kaina 
et al. 1990, Schwarz et al. 1995, Yao et al. 2000) and in cultures from metallothionein 
transgenic and knockout mice (Lazo et al. 1995, Kondo et al. 1995, Wang et al. 1999, 
Zheng et al. 1996) found similar results. Finally, a number of experiments in 
metallothionein transgenic and metallothionein knockout mice came to similar 
conclusion (Kang et al. 1997, Kang et al. 1999, Liu et al. 1995, Liu et al. 1998A, Liu et 
al. 1999A, Masters et al. 1994, Michalska and Choo 1993, Rofe et al. 1998). The results 
of several papers contradict the idea that metallothionein is universally protective, 
however (DiSilvestro et al. 1996, Itoh et al. 1997, Liu et al. 1999A, Minami et al. 1999). 

In some studies, metallothionein induction by zinc protected against the free 
radical generating hepatotoxin carbon tetrachloride. These conclusions were reached 
from in vitro studies, in vivo studies using nonspecific inducers of metallothionein, and 
studies using pharmacological injections of zinc. While injections of zinc are known to 
induce metallothionein and protect against oxidative stress in liver and cultured cells, it is 
not clear whether supplemental dietary zinc mimics the protective effects of parenterally 
administered zinc, and if so, whether the protection depends upon metallothionein 
production. Conversely, it is not known whether the effect of metallothionein induction 
on cytoprotection depends on the level of dietary zinc. Further, whether supplemental 
zinc and metallothionein expression act additively or synergistically is uncertain. 

Murine metallothionein knockout and metallothionein overexpressing models are 
more direct models of the effects of metallothionein expression on cytotoxicity. Also, 
zinc presented via the diet is more physiological than via injection. We determined 
whether metallothionein overexpressing mice or metallothionein knockout mice had 



43 

altered sensitivity to carbon tetrachloride compared to identically treated controls. We 
also determined whether supplemental dietary zinc reduced sensitivity to carbon 
tetrachloride in these genotypes. 

The results of the experiments explained herein confirm the importance of 
metallothionein expression in protection against oxidative stress, but bring into question 
the impact of supplemental zinc and/or elevated metallothionein expression in defense 
against oxidative stress in vivo. 

Materials and Methods 

Animals 

Metallothionein overexpressing mice (TG mice), C57BL/6 mice (CT mice), 
metallothionein knockout mice (KO mice) and 129/SvCPJ mice (CK mice) were used in 
these experiments. All experimental groups were age (8-1 1 wk) and sex matched. Mice 
were housed singly in stainless steel hanging cages with a 12 h light:dark cycle. During 
experiments, mice were given free access to deionized water and AIN-76A diets with 
adequate or supplemental zinc content (10 or 500 mg zinc/kg diet, respectively; Research 
Diets, New Brunswick, NJ). Care and treatment of the mice received approval of the 
University of Florida Institutional Care and Use Committee. 

Experimental Design 

TG, KO and control mice were acclimated to diet containing 10 mg zinc/kg (Zn 10 ) 
and deionized water for 7 d. Following this acclimation period, one half of the mice were 
switched to a diet containing 500 mg zinc/kg (Zn 500 ) for 3 d while the other half remained 
on the Zn 10 diet. After the third day of dietary treatment, mice were injected with carbon 






44 

tetrachloride (CC1 4 ) in corn oil (20 ul/kg bw, i.p.) or corn oil alone between 8 AM and 10 
AM, and animals were killed at h, 12 h, 24 h, and 48 h post dose. Since mice in the h 
group did not receive injections, data at this time point represent the effects of diet and 
genotype only. 

Food Intake and Body Weight 

Food intake was measured for the 3 d dietary treatment. Food intake intake is 
expressed as mg/g body weight. Body weight was measured at the onset of the 3 d 
dietary treatment. 

Analytical Methods 

109 Cd was measured using a Packard Cobra II gamma spectrometer equipped with 
a 3-inch crystal (Packard, Downers Grove, IL). Metallothionein protein was measured 
by the cadmium ( 109 Cd) binding assay (Eaton and Toal 1982). Total protein was 
measured by the method of Lowry et al. (1951). Serum zinc concentrations were 
measured by flame atomic absorption spectrophotometry (AAS) (Hempe and Cousins 
1989). Tissue zinc was measured as described previously (Dunn and Cousins 1989). 
Serum alanine aminotranferase (ALT) enzyme activity was measured 
spectrophotometrically as the formation of pyruvate from alanine and a-ketoglutarate 
using a commercial diagnostic kit (Sigma 505-P). The pyruvate formed is reacted with 
2,4-dinitrophyenylhydrazine, forming a 2,4-dinitrophenylhydrazone derivative that can 
be measured spectrophotometrically (A.max = 505 nm). The absorbance is converted to 
ALT activity units using a standard curve generated for pyruvate. 



45 

Total thiol groups were measured in liver homogenates spectrophotometrically 
after treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) (Jocelyn 1989). 
Reaction of sulfhydryl groups with DTNB generates a yellow chromophore (A.max = 412 
nm, extinction coefficient of e ■ 13100 M" 1 cm' 1 ). Nonprotein thiols were measured by 
the same technique after first removing protein thiols by TCA precipitation (5% TCA). 

Histological Analysis of Liver 

Sections of liver were fixed in 10% buffered formalin, embedded in paraffin, and 
stained with hematoxylin and eosin. These sections were analyzed visually for necrosis 
and other signs of hepatotoxicity (Khoo et al. 1996). Micrographs were obtained with a 
Zeiss Axiovert SI 00 microscope (Carl Zeiss, Thornwood, NY) fitted with a CCD camera 
for processing of digital images. 

Statistics 

Data were analyzed by ANOVA for a three way factorial design (2x2x2) to 
determine significant main effects and interactions using genotype, dietary zinc and 
oxidant treatment as independent variables (SAS, SAS Institute Inc. Cary, North 
Carolina). The Tukey-Kramer multiple comparison test was used to determine significant 
differences between specific groups. Serum ALT data were log transformed to obtain 
homogeneous variances. Significance was established at p < 0.05. 



46 



A 50 



Control 



Knockout 



g 40 

"o 
£ 

3 
u 

N 

£ 






B 



o 

900 



60 

I 600 

c 

o 

c 

N 

§3 300 
> 



■ 10+CO 
D 10+CC1 4 
A 500+CO 
A 500+CO, 








12 24 36 48 
Hours 



12 24 36 48 
Hours 



Figure 4-1. Indices of zinc homeostasis in metallothionein knockout (KO) and control 
(CK) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride (CC1 4 ) or corn oil 
(CO). The mice had been fed either adequate dietary zinc (Zn 10 ) or supplemental 
dietary zinc (Zn 500 ). (A) Serum zinc concentration (umol Zn/L serum). (B) Liver zinc 
(nmol/g liver). (C) Liver metallothionein (nmol/g liver). Data are presented as means 
± SE of n = 3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group. Statistical differences (p 
< 0.05) were determined by ANOVA for a three way factorial design, followed by the 
Tukey-Kramer multiple comparison test. 






47 

Results 
Food Intake 

Diet consumption was similar between adequate zinc (Zn I0 ) and supplemental 
zinc (Zn 500 ) groups, and was also similar between TG and CT groups (data not shown). 
Food consumption was statistically lower in KO than CK mice (p = 0.05), but the 
difference was unlikely to have been biologically significant (0.46 mg *g bw"' 3d" 1 vs 
0.48 mg * g bw" 1 3d 1 ). 

Zinc Status and Metabolism 

Serum zinc, liver zinc and liver metallothionein. were measured at h, 12 h, 24 h, 
and 48 h post-dose (Fig. 4-1 & 4-2). Since mice in the h group did not receive 
injections, data at this time point represent the effects of diet and genotype only. 

Knockout mice. The Zn 500 diet significantly increased serum zinc concentration 
in both genotypes, but to a greater extent in control (CK) mice (Fig. 4-1 A). The only 
significant effect of genotype was seen at 12 h after the CC1 4 treatment, when the serum 
zinc concentration increased by 80% in KO mice only. 

At Oh liver zinc content is significantly lower in KO mice than CK mice when fed the 
Zn 500 diet, but not the Zn 10 diet (Fig. 4- IB). Twelve hours later, CK mice fed the Zn 500 
diet and injected with CO (Zn 500 +CO) had 25-45% more liver zinc than other CK groups 
and significantly more (>100%) liver zinc than all KO groups. This effect may have been 
due to the stress of the injections themselves, but all mice received injections at this time 
point. The remaining CK groups had 35-90% more liver zinc than KO groups, but the 
differences were not statistically significance. At 24 h and 48 h there was significantly 
more liver zinc in CK 500 mice compared to all other genotype-diet combinations. The 



48 



only significant effect of CC1 4 treatment was at 24 h, when CCl 4 -treated mice had lower 
liver zinc values than CO treated mice. KO mice did not sequester zinc in the liver in 



Control 



Transgenic 



50 

I 40 

E 



g 

E 

E 
u 

00 



30 



20 



10 




g3600 

'35 

=3 2700 

E 



| 1800 

N 

■— 

J 900 



60 

o 

E 

f- 

u 

> 



275 

220 
165 

110 

55 




i i i i 







■ 


10+CO 






□ 


10+CC1 4 






▲ 


500+CO 






A 


500+CCl 4 


- -j 






A 




\ t! 




," ^r 


— 1 


1 \ 




, ' J ^---''^T 


- 










1 


1 


i i 








T * a 




















i 


£ 


x x 


t 




» 


— / 




** 








/ 












/ >r a 


I ^""---^A 








- jfi i 






*' \^i 


F 5 **«c--H 


3 


1 l l l 








\ 


/ 




/ 




\ 
\ 


/ 

/ -J 




L \ < 


x — i_ A 


k V 


Sj^\ 


3— ^Ofc 




i ^i 



12 24 36 48 
Hours 



12 24 36 48 
Hours 



Figure 4-2. Indices of zinc homeostasis in metallothionein transgenic (TG) and control 
(CT) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride (CC1 4 ) or corn oil 
(CO). The mice had been fed either adequate dietary zinc (Zn I0 ) or supplemental dietary 
zinc (Zn 500 ). (A) Serum zinc concentration (umol Zn/L serum). (B) Liver zinc (nmol/g 
liver). (C) Liver metallothionein (nmol/g liver). Data are presented as means ± SE of n = 
3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group. Statistical differences (P < 0.05) were 
determined by ANOVA for a three way factorial design, followed by the Tukey-Kramer 
multiple comparison test. 






49 

response to the Zn 500 diet, CC1 4 treatment, or combination of the treatments.Liver 
metallothionein levels depend on genotype and dietary zinc, and also are affected by 
oxidant treatment and the stress of the injection (Fig. 4-1C). These values roughly mirror 
hepatic zinc values. At h, 150% more metallothionein is detected in CK 500 compared to 
CK 10 mice. At 12 h after injection, CK 500 +CO mice had greater metallothionein values 
than other mice, and CK 500 mice had greater values than CK 10 mice. Results are similar at 
24 h. At 48 h, the metallothionein levels in the CK 500+CC14 group increased, and both CK 500 
groups have five fold greater metallothionein levels than CK, groups. As expected, KO 
mice did not express metallothionein. It appears that CC1 4 toxicity delayed the induction 
of metallothionein in these mice. 

Transgenic overexpressing mice. Zinc homeostasis was also altered by 
metallothionein expression. Serum zinc was 80-100% greater in CT mice fed the Zn 500 
diet than Zn 10 diet throughout the experiment (Fig. 4-2A). Serum zinc also rose in TG 
mice fed the Zn 500 diet, but -50% less than that found in CT mice. At 12 h, there was a 
trend toward decreasing serum zinc values after injection of vehicle alone in CT mice, 
and both CC1 4 and vehicle treated TG mice. In contrast, serum zinc values were 
significantly greater in CT+CC1 4 mice at this time point. This is similar to the serum zinc 
response of KO+CCL, mice. Throughout the time course of 48 h, TG mice exhibited 
better control over the serum zinc concentration, especially when consuming the Zn 500 
diet. Liver zinc was greater in TG 500 mice than CT 500 mice at h (Fig. 4-2B). At 12 h, 
however, TG 500 mice had greater zinc values than all other mice, and TG+CC1 4 mice had 
significantly greater liver zinc than all other genotype-oxidant combinations. At 24 h the 






50 

TG 500 mice had greater liver zinc values than all other diet/genotype combinations. Liver 
zinc declined in all groups between 24 h and 48 h, but remained greatest in TG 500 mice. 

As with CK and KO mice, liver metallothionein values mirrored the liver zinc 
values. TG 500 mice had more liver metallothionein at h than other diet-genotype 
combinations. Metallothionein was elevated in both genotypes at 12 h after CC1 4 
treatment. Metallothionein was elevated to its highest in all Zn 500 groups at 24 h after 
injection, and were greatest when the injection contained CC1 4 . At 48 h liver 
metallothionein declined in all groups, but the TG 500 and TG CC14 groups still had slightly 
more metallothionein than all other genotype-diet and genotype-oxidant combinations, 
respectively. Interestingly, CC1 4 alone had a minimal effect on liver metallothionein in 
CT or TG mice unless combined with the Zn 500 diet. 



S 3 

I § 

S'5 
oo -a 



3- 



2- 



Control 



IJ" 



■ 10+CO 
□ I0+CC1 4 
A 500+CO 
S A 500+CCL 



\ \ 




VI 

=1 



j_ 



12 24 36 
Hours 



48 



_L 



Knockout 



ti 



*\, 




_L 



± 



12 24 36 48 
Hours 



Figure 4-3. Serum alanine aminotransferase activity of metallothionein knockout (KO) 
and control (CK) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride (CC1 4 ) or 
corn oil (CO). The mice had been fed either adequate dietary zinc (Zn 10 ) or supplemental 
dietary zinc (Zn 500 ). These activities were measured spectrophotometrically and 
expressed as log activity units/mL serum. Data are presented as means ± SE of three (0 h) 
or 4-7 mice/group (12, 24, and 48 h). Data are presented as means ± SE of n = 3 (0 h) or 
n = 4-7 (12, 24, and 48 h) mice/group. Statistical differences (p < 0.05) were determined 
by ANOVA for a three way factorial design, followed by the Tukey-Kramer multiple 
comparison test. 



51 

Hepatotoxicity and Oxidative Stress 

Hepatotoxicity was assessed by measuring serum alanine aminotransferase 
enzyme (ALT) activity, and by histological analysis of liver sections for signs of damage 
and necrosis. Measurement of liver nonprotein thiols (a pool comprised mainly of GSH 
molecules) and liver total thiols served as measures of oxidative stress. 

Knockout Mice. All mice had similar and normal serum ALT activity levels at 
h (Fig. 4-3). At 12 h all mice had significantly elevated serum ALT activity, but the mean 
ALT level (actual ALT units) was 6-12 times greater in KO mice than in CK mice. At 24 
h and 48 h after CC1 4 , the ALT levels had declined, but were still significantly greater in 
CC1 4 treated mice. There were no genotype effects at the 24 h and 48 h points, and no 
effects of dietary zinc at any time point. 

Hematoxylin- and eosin-stained liver sections were similar in all groups injected 
with corn oil (data not shown). Consistent with serum ALT activities, at 12 h there was 
significantly more liver necrosis in KO mice (Fig. 4-4B) compared to CK mice (Fig. 4- 
4A). The perivenous regions of the KO liver sections displayed coagulation necrosis. 
Cells were less eosinophilic, displayed a general loss of morphology, and many contained 
pyknotic nuclei. Hepatic sinusoids were collapsed and the hepatic plate arrangement was 
disrupted. In contrast, little necrosis was visible in CK liver sections. These differences 
were not seen at 24 h and 48 h, however. There were no differences between dietary 
groups. 

Oxidative stress was measured as nonprotein thiol levels (Fig. 4-5 A). Nonprotein 
thiols (NPT) are a thiol pool made up largely of glutathione, and have been shown to 
decrease in instances of oxidative stress (Powers et al. 1998). Although glutathione 



52 



concentrations are subject to circadian rhythms, this variable was controlled for by 
inclusion of corn oil-treated mice for all combinations of genotype and dietary zinc. 
Interestingly, NPT levels at h tended to be lower in Zn 500 groups than Zn 10 groups, but 
the difference was not quite significant (p = 0.06). At 12 h post injection, there was a 




Figure 4-4. Light micrographs (200X magnification) of hematoxylin- and eosin-stained 
liver sections from metallothionein knockout (KO) or control (CK) mice 12 h after 
injection of carbon tetrachloride (CC1 4 ). (A) CK mouse; (B) KO mouse. All mice were 
fed diets contaoining 10 mg Zn/kg diet (Zn l0 ). More severe coagulation necrosis was seen 
around the central vein (cv = central vein) in KO mice (Panel B) compared to CK mice 
(Panel A). Results were similar in female mice and mice fed diets containing 500 mg 
Zn/kg diet (Zn 500 ). No histological changes due to genotype or diet were noted in mice 
given an injection of the corn oil vehicle. Bar = 100 urn. 



53 



general depression of NPT, but no differences among groups. At 24 h NPT levels were 
significantly lower in the KO+CCl 4 groups, while KO+CO groups returned to normal. 
Interestingly, at 48 h CC1 4 treated mice have significantly more NPTs in liver than CO 
treated mice. Total thiols (TT) are a measure of both protein and nonprotein thiols 
combined, including the thiols of metallothionein. There was a trend toward greater TT 
levels in KO mice than CK mice at h (Fig. 4-5B). At 12 h post treatment, however, 



Control 



Knockout 



400 



300- 



> 
c 3 



H ,£? 200 



o 

& E 
o S 



100 




12- 



j= — 

f- 60 

— ;a- 

CS Q 



6- 



12 24 
Hours 



36 48 



x 




r /XN^* 


U-.^./V 


f ••3t- » 


- J_ ■**■ 


1 1 1 1 ill 



i^ 5 ^ 




i 


■ 10+CO 
□ 10+CC1 4 
A 500+CO 
A 500+CCl 4 

1 i ! 




12 24 36 48 
Hours 



Figure 4-5. Indices of liver thiol homeostasis and oxidative stress of metallothionein 
knockout (KO) and control (CK) mice 0, 12, 24, or 48 h after injection of carbon 
tetrachloride (CC1 4 ) or corn oil (CO). The mice had been fed either adequate dietary zinc 
(Zn 10 ) or supplemental dietary zinc (Zn 500 ). (A) Liver nonprotein thiols (nmol/g liver) 
were measured spectrophotometrically. Protein thiols had been removed by TCA 
precipitation. (B) Liver total thiols (umol/g liver) were measured spectrophotometrically. 
Data are presented as means ± SE of n = 3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group. 
Statistical differences (p < 0.05) were determined by ANOVA for a three way factorial 
design, followed by the Tukey-Kramer multiple comparison test. 



54 

liver TT levels in KO mice drop, and are significantly lower than in CK mice. At 24 h 
and 48 h no differences were detected. 

Transgenic overexpressing mice. Serum ALT activity was similar and normal at 
h in all groups (Fig. 4-6). Serum ALT rose sharply in all groups treated with CC1 4 at 12 
h, and remained elevated through 48 h. There were no differences due to genotype. ALT 
was slightly greater in Zn 500 treated mice than Zn 10 treated mice at 48 h, however. Of 
note, ALT activities do not return to basal levels, as was observed with CK and KO mice 
(Fig. 4-3) 

Histological analysis of hematoxylin- and eosin-stained liver sections revealed 
significant necrosis in all CCl 4 -treated mice, but there were no significant differences 
between genotype (Fig. 4-7 A,B). There was significant lymphocyte infiltration in the 



Control 



Transgenic 



-7 3 



il 

en ■&■ 

2 - > 




oL^ 



± 



_i_ 



12 24 36 48 
Hours 







- i 


*■-----. 




" 1 






l> 


■E 


— §• 




" -r- 




fJL. 




HS 






-S tr — ■ 






1 1 1 1 





12 24 36 48 
Hours 



Figure 4-6. Serum alanine aminotransferase activity of metallothionein transgenic 
(TG) and control (CT) mice 0, 12, 24, or 48 h after injection of carbon tetrachloride 
(CC1 4 ) or corn oil (CO). The mice had been fed either adequate dietary zinc (Zn 10 ) or 
supplemental dietary zinc (Zn 500 ). These activities were measured 
spectrophotometrically and expressed as log activity units/mL serum. Data are 
presented as means ± SE of three (0 h) or 4-7 mice/group (12, 24, and 48 h). Data are 
presented as means ± SE of n = 3 (0 h) or n = 4-7 (12, 24, and 48 h) mice/group. 
Statistical differences (p < 0.05) were determined by ANOVA for a three way 
factorial design, followed by the Tukey-Kramer multiple comparison test. 



55 



.A 



HH 



.cv- 1 



cv 



• 



C- 









' ■ " .-t CV .' . 



»•• " 







• - 




Figure 4-7. Light micrographs (200X magnification) of hematoxylin- and eosin-stained- 
liver sections from metallothionein transgenic (TG) or control (CT) mice 12 h after 
injection of carbon tetrachloride (CC1 4 ). (A) CT mouse fed the Zn l0 diet; (B) TG mouse 
fed the Zn I0 diet; (C) CT mouse fed the Zn 500 diet; (D) TG mouse fed the Zn 500 diet; (E) 
enlargement of the perivenous region from figure 4-7D. Significant lymphocyte 
infiltration is seen in the area directly surrounding the central vein (cv) of mice of both 
genotypes fed the Zn 500 diet after receiving CC1 4 (C-E). Results were similar in female 
mice. No histological changes were related to genotype were noted in mcie given an 
injection of corn oil vehicle and fed either diet. Bar = lOOum (A-D) or 25 urn (E). 



56 

area directly surrounding the central vein of ce of both genotypes fed the Zn 500 diet (Fig. 
4-7C-E). 

There were no significant differences in NPT between TG and CT mouse livers at 
h (Fig. 4-8A). Twelve hours after injection, the NPT levels were higher in CCl 4 -treated 
mice. This relationship reversed at 24 h, and NPT levels were greatest in CT+CO mice. 
Similar results were seen at 48 h. 

Total thiol levels were similar in all mouse groups at h (Fig. 4-8B). At 12 h 
there is a trend toward greater TT levels in TG 500+CC14 mice compared to others. This is 
likely due to induction of hepatic metallothionein. At 24 h, CCl 4 -treated mice had 
significantly greater TT levels than CO treated mice. Also, TG mice had significantly 
greater TT levels than CT mice. These significances disappeared at 48 h, but there is still 
a trend toward greater TT levels in TG CCW mice compared to TG co mice. Since the thiols 
of metallothionein would be included in the TT measurement, and metallothionein 
induction was greatest at 24 h and 48 h, metallothionein induction alone may have 
accounted for the increased TT in TG mice at these time points. These also are the time 
points of NPT suppression (Fig. 4-8 A). 

Discussion 

We examined the effects of supplemental dietary zinc in combination with 
different levels of metallothionein gene expression on susceptibility to oxidative stress in 
vivo. Previous research showed that supplemental zinc protected rat hepatocytes cultures 
from various cytotoxic agents (Coppen et al. 1988, Schroeder and Cousins 1990). Zinc 
treatment increased metallothionein gene expression, and it was suggested that 
metallothionein protein was the mediator of the protection. Similar results were seen 



57 

when metallothionein expression was elevated by interleukin-6 and dexamethasone 
(Schroeder and Cousins 1990). In this experiment, we used KO and TG mice as models 
of different levels of metallothionein gene expression (Masters et al. 1994, Palmiter et al. 
1993). By using two zinc intake levels, we were able to generate different hepatic zinc 



Control 



450 



300 



Transgenic 



f- > 




a — 




'5 -5» 




— — 




2 ° 




D. S 




o ■— ' 


150 


Z 






24 



18- 



1 1 



■ 


10+CO 


□ 


10+CC1 4 


*v * * "ffi A 


500+CO 


- J\- - -2^ . A 


500+CCl 4 


^^A * » 






^A » \ 






v x x 1 




i* ■ — — jt~l 


SL -ii"' 




1 I I 


I i 




J_ 



_L 




_L 



12 24 36 48 
Hours 



12 24 36 
Hours 



48 



Figure 4-8. Indices of liver thiol homeostasis and oxidative stress of metallothionein 
transgenic (TG) and control (CT) mice 0, 12, 24, or 48 h after injection of carbon 
tetrachloride (CC1 4 ) or corn oil (CO). The mice had been fed either adequate dietary 
zinc (Zn l0 ) or supplemental dietary zinc (Zn 500 ). (A) Liver nonprotein thiols (nmol/g 
liver) were measured spectrophotometrically. Protein thiols had been removed by 
TCA precipitation. (B) Liver total thiols (umol/g liver) were measured 
spectrophotometrically. Data are presented as means ± SE of n = 3 (0 h) or n = 4-7 
(12, 24, and 48 h) mice/group. Statistical differences (p < 0.05) were determined by 
ANOVA for a three way factorial design, followed by the Tukey-Kramer multiple 
comparison test. 



58 

and metallothionein levels to examine their effects on the toxicity of the hepatotoxin 
CC1 4 . CCI4 is metabolized to the trichloromethyl radical by the enzyme cytochrome 
P4502E1 in the perivenous region of the liver lobule (McGregor and Lang 1996, Sipes et 
al. 1990; Wong et al. 1998). As a result, the perivenous region is most affected by this 
radical species, which causes lipid peroxidation and inactivation of enzymes such as the 
cytochrome P450 enzymes (McGregor and Lang 1996). 

In several studies zinc pretreatment protected against CCl 4 -induced hepatotoxicity 
in vivo (Cagen and Klaassen 1979, Chvapil et al. 1973, Dhawan and Goel 1995, Liu et al. 
1998). This effect is only seen when copious amounts of dietary zinc (1000 mg/kg diet) 
or parenteral zinc are given, however. For example, consumption of diets containing 300 
mg Zn/kg, twenty fold the requirement for the rat, did not protect against CCl 4 -induced 
hepatotoxicity in rats (Khoo et al. 1996). A proposed mechanism for the protective effect 
of pharmacological doses of zinc is that zinc induces metallothionein, and that 
metallothionein is the real mediator of the hepatoprotection. Metallothionein has been 
shown to covalently bind CC1 4 metabolites and decrease the amount of these metabolites 
bound to other cellular proteins (as reviewed in Klaassen and Liu 1998). In this way 
metallothionein may prevent CC1 4 from reaching some of its cellular targets. Although 
this might explain some of the zinc related protection, large parenteral zinc doses also 
protect against CC1 4 toxicity in the absence of metallothionein expression (Itoh et al. 
1997). This might be related to suppression of CC1 4 bioactivation, since supplementation 
has been shown to inhibit the activity of some cytochrome P450 enzymes (Bray et al. 
1986, Coppenetal. 1988). 



59 

Since metallothionein expression has been inversely related to damage after an 
oxidative insult, we expected that the damage in mouse livers in this experiment would 
be inversely proportional to metallothionein expression (i.e., TG < CT and CK < KO). In 
support of this, ehanced susceptibility of KO mice to CC1 4 toxicity was reported recently 
(Liu et al. 1998B). Also, we assumed that mice consuming the Zn 500 diet would be 
protected compared to those eating the Zn l0 diet since supplementation would induce 
more metallothionein protein and result in greater cellular zinc accumulation. Finally, 
since metallothionein expression was thought to be key to zinc-related cytoprotection, we 
expected no protection against hepatotoxicity by zinc supplementation in KO mice. 

In support of our hypothesis we saw greater hepatotoxicity in KO mice compared 
to CK mice (6-12 fold higher ALT values) at the 12 h time point. At later time points, 
however, toxicity seemed equivalent in these genotypes. This is in spite of the fact that 
KO mice had no metallothionein present in liver and were unable to sequester additional 
hepatic zinc. Also, TG and CT mice did not differ in the level of hepatotoxicity produced 
despite huge differences in hepatic metallothionein and zinc. Further, the nonprotein thiol 
levels were not dramatically altered during the experiment, similar to results from 
experiments with acetominophen in KO mice (Liu et al. 1999 A). 

To our knowledge, this is the first report where the effect of supplemental dietary 
zinc on CCl 4 -induced hepatotoxicity in mice was examined. Also, this is the first 
assessment of the combination of supplemental dietary zinc and toxicity of any kind in 
metallothionein overexpressing mice and metallothionein knockout mice. We show for 
the first time using these models that neither supplemental dietary zinc nor 
metallothionein overexpression alone protected against CCl 4 -induced hepatotoxicity in 



60 

mice. Further, no combination of metallothionein gene expression and either adequate or 
supplemental dietary zinc provided protection, even though the levels of liver zinc and 
liver metallothionein varied over a large range among groups. 

These results argue against a direct antioxidant role for MT against CC1 4 toxicity, 
since antioxidant protection against hepatotoxicity would likely be dose dependent 
(Tirmenstein et al. 1997, Yao et al. 1994). Instead, the data fit better in a plateau model, 
where metallothionein expression was important up to a point (< the level in CT and CK 
mice), but beyond this point further expression is not useful. This is more in line with a 
metallothionein-specific function, such as regulation of tissue zinc accumulation and/or 
intracellular zinc trafficking. Specifically, KO mice might be less protected against CC1 4 
than CK mice because they are unable to regulate zinc homeostasis appropriately (Coyle 
et al. 1995, Davis et al. 1998, Philcox et al. 1995). 

The most marked differences between genotypes in this experiment were 
alterations in zinc metabolism. In both the TG and the KO experiments, the animals with 
the lowest metallothionein expression displayed the weakest control over serum zinc 
levels 12 h after CC1 4 treatment. In fact, serum zinc rose in KO mice and declined in TG 
mice. This is coincident with induction of hepatic metallothionein and elevation of 
hepatic zinc in all but the KO mice at this time point. This is also coincident with greater 
hepatotoxicity in KO mice. 

Metal lothionein's role in maintaining appropriate hepatic zinc levels might be 
especially important under conditions of stress, including oxidative stress. 
Metallothionein is induced during the acute phase response and during hepatic 
regeneration, and is required for normal hepatic regeneration after partial hepatectomy 



61 

(Arora et al. 1998, Ohtake 1978). Also, zinc can be mobilized from metallothionein by a 
number of oxidants or shifts in glutathione redox status (Berendji et al. 1997, Fliss and 
Menard 1992, Tatsumi and Fliss 1994). These may be mechanisms for mobilization of 
intracellular zinc during oxidative stress (Maret 1995). The end result may be enhanced 
transfer of zinc from metallothionein to zinc-dependent proteins (Jiang et al. 1998). Zinc 
mobilization may also affect gene expression. 

If metallothionein acts as a zinc-donating molecule during oxidative stress, KO 
mice might not be able to keep up with the demand for zinc incorporation into zinc- 
dependent proteins. As a result, the level of functioning zinc-dependent proteins 
produced during oxidative stress may not be sufficient in KO mice, and may explain why 
KO mice were more adversely affected than CK mice. On the other hand, TG mouse 
cells have more zinc available for donation to zinc-dependent proteins, but they may not 
be able to produce those proteins rapidly enough to take advantage of the excess zinc. In 
this case, the level of MT protein produced in CT mice is both necessary and sufficient, 
and TG mice would not be better protected. 

While other explanations for the results of this experiment exist, we can rule out 
several. First of all, the results of this experiment are not likely affected by differences in 
other antioxidants in these mice since the levels of other antioxidant enzymes and 
molecules are reported to be similar between genotypes (Iszard et al. 1995, Kang et al. 

1997, Lazo et al. 1995, Liu et al. 1999, Rofe et al. 1998, Wang et al. 1999, Wu and Kang 

1998, Zheng et al. 1996). Also, bioactivation of CC1 4 should be similar between 
genotypes since the activity of cytochrome P4502E1 is similar in TG and KO mouse 
livers to their respective controls (Iszard et al. 1995, Itoh et al. 1997, Rofe et al. 1998). 



62 

Since these results are in opposition to some experiments done in rats, we cannot exclude 
species difference as a confounding variable. However, it seems unlikely that a process 
as basic as radical scavenging (antioxidant action), a simple oxidation-reduction reaction, 
would differ between two rodent species. It should be noted that different strains of mice 
were used in these experiments, and, therefore, the results are not likely due to 
peculiarities of any individual inbred strain. However, we cannot rule out the possibility 
that knockout mice are better protected than we expected due to some adaptive 
mechanism(s). For instance, altered gene expression has been reported in KO mice 
(Kimura et al. 2000). 

Two other research groups have published reports of CCl 4 -induced hepatotoxicity 
in KO mice, but no reports have been forwarded for TG mice (Itoh et al. 1997, Liu et al. 
1998B). Those KO studies were designed to differentiate the hepatoprotective effects of 
exogenous compounds (sakuraso-saponin and oleanic acid) from their ability to induce 
metallothionein synthesis. Both groups used the same KO mouse model, injected 50 ul 
CCl 4 /kg bw, and reported results for 24 h post-dose. Itoh and coworkers found no 
difference in hepatotoxicity between genotypes as measured by plasma GOT activity. Liu 
and coworkers found greater damage in KO mice as measured by serum ALT, serum 
SDH, and histological analysis of hematoxylin- and eosin-stained liver sections. Itoh and 
coworkers injected the dose subcutaneously, while Liu and coworkers injected the dose 
intraperitonealy. Slower uptake of CC1 4 from the subcutaneous injections of Itoh and 
coworkers might explain why no toxicity was seen at the 24h time course in their study. 
Serum ALT values and histological analysis from the 12 h time point in this report match 
well with the results of Liu and coworkers at 24 h. The differences in the time course of 



63 

hepatotoxcity between that experiment and ours may have been due to the smaller dose 
(20 ul CCl 4 /kg bw) used in our experiments. 

The lack of protection against oxidative stress by MT overexpression in this 
experiment is in line with results from several other experiments that used models of 
metallothionein gene overexpression. Early studies using transfection of the human MT- 
2a gene into Chinese hampster ovary Kl-2 cells and several of tumor cells lines found no 
resistance against free radical generating agents (Kaina et al. 1990, Kelley et al. 1988). 
Further, TG mice were not resistant to adriamycin cardiotoxicity or y-irradiation 
(DiSilvestro et al. 1996, Liu et al. 1999B). The existing evidence that metallothionein 
overexpression alone protects against oxidative stress was found in transformed cells and 
sheep pulmonary artery endothelial cells (SPAEC) transfected with metallothionein, and 
with a second TG mouse strain that only overexpresses metallothionein in the heart. In 
the former case, transfection of NIH 3T3 cells with MT-1 protected against nitric oxide 
induced cytotoxicity (Schwarz et al. 1995). Viable cell determinations were not made 
until 6 to 7 days after nitric oxide treatment, however, so it could be argued that the 
difference in the number of cells remaining at that time was due to improved cell 
recovery instead of radical scavenging. Protection against hyperoxia and tertiary-butyl 
hydroperoxide was also seen in SPAEC transfected with mouse or human MT genes, but 
the determination of viable cell numbers were not performed until 1-2 days after oxidant 
exposure was initiated (Pitt et al. 1997). Again, it is difficult to separate the contributions 
of antioxidation and cell recovery to cell survivial. 

In the case of the heart-specific metallothionein overexpressing TG mouse strain, 
there is both in vivo and in vitro evidence that metallothionein protected against 



64 

oxidative stress, including ischemia reperfusion, hydrogen peroxide, and doxorubicin 
treatment (Kang et al. 1997, Kang et al. 1999, Wang et al. 1999, Wu and Kang 1998). 
While this provides convincing evidence for an antioxidant role for metallothionein in 
cardioprotection, it should be noted that a very high level of overexpression (10-130- 
fold) is needed to see these effects, as lower levels of overexpression (3-fold) didn't 
protect against adriamycin cardiotoxicity (DiSilvestro et al 1996). Further, mouse heart, 
an organ that metallothionein is not normally abundant in, has a much weaker antioxidant 
capacity than mouse liver (as reviewed in Kang 1999). So while this model confirms that 
metallothionein overexpression can protect mouse heart against oxidative stress, the 
effects may be small since they were unmasked only when metallothionein expression 
was astronomically high, in an organ that doesn't normally produce much 
metallothionein, yet is highly susceptible to oxidative stress. Therefore, it cannot be 
assumed that the same results would be seen in the liver. 

The lack of protection against CC1 4 hepatotoxicity by zinc supplementation (500 
mg/kg diet) in any of the genotypes used in this experiment strongly suggests that the 
required dietary zinc level for mice (10 mg/kg diet) provides as much protection as is 
possible by dietary zinc. It also suggests that the hepatoprotective effects associated with 
zinc injection are not readily reproduced with dietary zinc. Combined with data from zinc 
deficiency studies we see a plateau affect of dietary zinc against oxidative stress in the 
rodent model, just as we do with metallothionein. Zinc-deficient diets render rodents 
more susceptible to oxidative stress, zinc-adequate diets alleviate this condition, but 
supplemental dietary zinc provides no further protection. 



65 



The results of the experiments explained above confirm the importance of 
metallothionein expression in protection against oxidative stress, but bring into question 
the impact of supplemental zinc and/or elevated metallothionein expression in defense 
against oxidative stress in mouse liver in vivo. Further, the protection against oxidative 
stress appears to correlate with changes in zinc metabolism. 



CHAPTER 5 

EFFECTS OF METALLOTHIONEIN GENE EXPRESSION AND SUPPLEMENTAL 

ZINC IN PROTECTION AGAINST OXIDATIVE STRESS IN PRIMARY 

HEPATOCYTE CULTURES FROM METALLOTHIONEIN TRANSGENIC AND 

METALLOTHIONEIN KNOCKOUT MICE 

Introduction 

As indicated in the previous chapter, metallothionein and zinc are implicated in 
cellular antioxidant defense. In the previous experiment with metallothionein knockout 
mice (KO) we found that metallothionein expression protected against carbon 
tetrachloride-induced oxidative stress, but metallothionein overexpression in transgenic 
mice (TG) did not provide further protection. Further, mice fed diets with supplemental 
zinc (500 mg Zn/kg diet) were not protected compared to mice fed only the required zinc 
intake (10 mg Zn/kg diet). These results are inconsistent with direct antioxidant activity 
of metallothionein induction or supplemental zinc. To more directly assess the cellular 
antioxidant functions of supplemental zinc and metallothionein, We studied the effects of 
metallothionein expression and moderate zinc supplementation on tertiary-butyl 
hydroperoxide-induced cytotoxicity in primary hepatocyte cultures from TG and KO 
mice. Specifically, hepatocytes from metallothionein knockout mice can be used to 
determine if zinc acts in cytoprotection independent of metallothionein by examining 
whether these cells had altered sensitivity to tertiary-butyl hydroperoxide. We also 
determined whether treating cells with zinc and/or dexamethasone and interleukin-6, all 
of which induce metallothionein expression, influences sensitivity to tertiary-butyl 



66 



67 

hydroperoxide in these genotypes. The results of this study provide further evidence that 
metallothionein and supplemental zinc do not act directly as antioxidants. 

Materials and Methods 
Animal Model 

Metallothionein knockout and metallothionein transgenic mice used in this study 
were bred in house using founder mice purchased from The Jackson Laboratory, Bar 
Harbor, ME. The metallothionein overexpressing mice (designated TG mice) were 
originally generated in C57BL/6 mice crossed with SJL mice (Palmiter et al. 1993). 
Backcrossing against C57BL/6 mice permit the use of C57BL/6 mice as controls 
(designated CT mice). The metallothionein knockout mice (designated KO mice) were 
generated in 129/SvCPJ mice crossed with C57BL/6 mice (Masters et al. 1994). These 
mice were maintained on a 129/SvCPJ background. 129S3/SvImJ mice served as controls 
(designated CK mice). All experiments used 7-11 wk old female mice. Mice were housed 
in plastic box cages with a 12 h light:dark cycle. The mice were given access to tap water 
and a standard rodent diet (Harlan Teklad 8604, Madison, WI) until they were used for 
hepatocyte preparations. Care and treatment of the mice received approval of the 
University of Florida Institutional Care and Use Committee. 

Hepatocyte Isolation 

All liver perfusions began between 8 AM and noon. In all experiments 
hepatocytes were collected from mice of both genotypes (KO and CK or TG and CT) on 
the same day. Mice were anaesthetized with sodium pentabarbital (60 mg/kg i.p.). 
Hepatocytes were isolated using a two-step perfusion technique run retrograde from the 



68 

inferior vena cava, with perfusate allowed to flow out of the portal vein without 
recirculation (Schroeder and Cousins 1991, Renton et al. 1978). Livers were first 
perfused with a Ca-free buffered solution (8-10 mL/min), followed by a buffered solution 
containing collagenase. After perfusion the liver was aseptically transferred to a sterile 
cell culture hood. The liver was disrupted and the cells were liberated using a cell 
scraper. 

Hepatocyte Culture 

The hepatocytes were suspended in wash medium (Williams Medium E with 10 
mmol/L HEPES buffer and 10 mmol/L TES buffer) followed by centrifugation (50 x g 
for 3 min) to separate viable cells from debris. The wash and centrifugation steps were 
repeated two times. The final cell pellet was resuspended in culture medium (Williams 
Medium E supplemented with 10% FBS, 100 nmol/L insulin, 100 units/mL penicillin, 
100 ug/mL streptomycin). Cell number and viability were determined using a 
hemacytometer and trypan blue exclusion. Preparations with greater than 85% viability 
were used for experiments. Cells were added to type I collagen-coated 35 mm tissue 
culture plates (0.5x 10 6 cells/plate in 2 mL medium) and allowed to attach (37°C, 5% 
C0 2 ). After a 2-3 h attachment period the culture medium was removed and replaced 
with 1 mL of one of four different medium combinations: medium containing 4 umol 
zinc/L, medium with added zinc (32 umol/L), medium with dexamethasone (1 umol/L) 
and interleukin-6 (100 units/mL), and medium with added zinc, dexamethasone and 
interleukin-6. Cells were maintained in these culture conditions for 1 8-22 h prior to 
addition of tertiary-butyl hydroperoxide (TBH). Carbon tetrachloride was also used in 



69 

some experiments, but its nonpolar nature and volatility led to problems with 
reproducibility within and between experiments. 
Cytotoxicity assays 

After the culture period was complete the medium was again removed and 
replaced with medium containing to 500 umol/L TBH for 30 to 150 min. At the end of 
TBH treatment, cells and medium were prepared for determination of lactate 
dehydrogenase (LDH) activity. Briefly, medium was centrifuged (13000 x g) to remove 
unattached cells and debris, and then stored at -20°C for up to 48 h before assay. Cells 
were removed in 1% triton X-100, disrupted by repeated passage through 200 uL pipette 
tips, and centrifuged (13000 x g). The supernatant was diluted with 4 volumes of Tris 
buffer (100 mmol/L 2-amino-2-(hydroxymethyl)-l,3-propanediol, pH 7.5) and stored at - 
20°C for up to 48 h before assay. LDH activity was assayed spectrophotometrically 
using a commercial kit (Sigma LD-L) by incubating aliquots of medium or cell extracts 
(125 uL) with reagent solution (375 uL) containing lactate (50 mmol/L) and NAD (7 
mmol/L) in buffer (pH 8.9). LDH in the sample catalyzes the reaction of lactate and 
NAD, resulting in the production of pyruvate and NADH. Formation of NADH results in 
an increase in absorbance at 340 nm. The rate of increase in absorbance is directly 
proportional to LDH activity in the sample. The percent LDH leakage is calculated as the 
LDH activity of the medium as a percentage of the sum of the LDH activities of the 
medium + the cells (Jauregui et al. 1981). 

Cell viability was also determined by the ability of live cells to convert 3-[4,5- 
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) to a colored 
product (Denizot and Lang 1986). In these experiments, cells were cultured on collagen 



70 

coated 96 well plates (3 x 10 4 cells/well in 200 uL WME) and treated as described above 
for 35 mm plates. At the end of the culture period the medium was replaced with medium 
containing 1-50 umol/L TBH and incubated for 120 min. This lower range of TBH 
concentrations was used in these experiments to correct for the greater ratio of medium 
volume-to-cell number. Medium containing TBH was then replaced with serum-free 
culture medium containing MTT (2.4 umol/L) for 3 h. MTT is a tetrazolium dye that is 
converted to an insoluble purple formazan through cleavage of the tetrazolium ring by 
dehydrogenase enzymes of live cells. Cultures were washed and then solubilized in 100 
uL acidic isopropanol (40 umol/L HC1 in absolute isopropanol). After shaking the wells 
were analyzed (A 560 -A 650 ). Data are presented as percent of activity of untreated cultures. 

Analytical Methods 

For intracellular glutathione analysis cells were homogenized in 1% (39 mmol/L) 
sulfosalicylic acid (2-hydroxy-5-sulfobenzoic acid; SSA) and placed on ice (> 20 min) to 
precipitate proteins, which were then removed by centrifugation (13000 x g). The 
supernatants were frozen overnight (-20°C). Total glutathione (oxidized + reduced) was 
measured spectrophotometrically by the glutathione (GSH) reductase-recycling assay 
using a microtiter plate reader (Baker et al. 1990). Briefly, supernatants were diluted with 
buffer (100 mmol/L sodium phosphate, 1 mmol/L EDTA, pH 7.5) and reacted with 5,5'- 
dithiobis-(2-nitrobenzoic acid) (DTNB; 150 umol/L) in the presence of NADPH (100 
umol/L) and GSH reductase (1.0 units/mL). Reaction of GSH with DTNB generates 
oxidized glutathione (GSSG) and the highly colored 5-thio-2-nitrobenzoic acid anion, 
which can be measured at A. = 410 nm. GSH is regenerated from GSSG by glutathione 
reductase (using reducing equivalents from NADPH), which allows the reaction between 



71 



GSH and DTNB to continue at a linear rate. GSH concentration is determined from a 
standard curve using GSSG. 109 Cd (1.35 GBq/nmol; Isotope Product Laboratories, 
Burbank, CA) was measured using a Packard Cobra II gamma spectrometer (Packard, 
Downers Grove, IL). Metallothionein protein content of the cells was measured prior to 
treatment with oxidant by the cadmium ( 109 Cd) binding assay (Eaton and Toal 1982). 
Briefly, cells were lysed in 10 mmol/L Tris buffer containing protease inhibitors (0.1 
mmol/L phenylmethylsulfonylfluoride, 1.2 u,mol/L leupeptin and 1.5 umol/L pepstatin 
A). Homogenates were centrifuged (10000 x g), the supernatant was boiled and 
centrifuged again (10000 x g), and the resulting supernatant was incubated with 109 Cd. 
Unbound l09 Cd was removed using hemoglobin. 109 Cd bound to metallothionein was 
measured by y-counting, and converted to moles of metallothionein (Davis et al. 1998). 
Cell zinc was measured by atomic absorption spectrophotometry after cells were 
solubilized in an aqueous solution of sodium dodecyl sulfate (7 umol/L) and sodium 
hydroxide (400 mmol/L) as previously described (Schroeder and Cousins 1991). Total 
protein was measured by the method of Lowry et al. (1951). 

Statistics 

Data were analyzed by ANOVA for a three way factorial design (2x2x2) to 
determine significant main effects and interactions among genotype, medium zinc and 
dexamethasone/Il-6 treatment (SAS, SAS Institute Inc. Cary, North Carolina). The 
Tukey-Kramer post hoc test was used to determine significant differences between 
specific groups when interactions were significant (p < 0.05). 



72 

Results 

Hepatocyte metallothionein expression was directly related to MT genotype and 
was inducible by zinc treatment, dexamethasone and 11-6 (dex/Il-6) treatment, and the 
combination of these treatments in all but KO cells (Fig. 5-1). Also, induction by these 
treatments was greater in TG cells than either control strain. Further, metallothionein 
induction by the combination of zinc treatment and dex/Il-6 treatment was synergistic. 

Cell zinc concentrations were lower in KO cultures than CK cultures under all 
treatment conditions (Table 5-1). As with metallothionein, cell zinc was increased in CK 
cultures in response to zinc treatment and dex/Il-6 treatment, and was increased 
synergistically by these two treatments combined. Cell zinc was not affected by these 
treatments in KO cultures, however. 

Lactate dehydrogenase (LDH) enzyme leakage was used as a measure of 
cytotoxicity. Leakage was not greatly different between CK and KO cultures after 
treatment with TBH (350 umol/L or 450 umol/L) when cells were not previously 
exposed to MT inducers (Fig. 5-2A). Zinc pretreatment modestly elevated LDH 
leakagefrom CK mouse hepatocytes treated with 450 umol/L TBH, but did not alter 
toxicity in KO cultures. Treatment with dex/Il-6 slightly increased LDH leakage in KO 
cells in response to 450 umol/L TBH, but increased toxicity in CK cells by three fold. It 
should be noted that neither zinc treatment nor dex/Il-6 treatment increased LDH leakage 
in the absence of TBH. The combination of zinc treatment with dex/Il-6 treatment was 
associated with a similar level of TBH- induced toxicity as dex/IL-6 treatment alone. 

Similar trends were seen in experiments with hepatocytes from TG and CT mice. 
(Fig. 5-2B). LDH leakage was already greater in TG cultures than CT cultures without 



73 

exposure to metallothionein-inducing agents. Pretreatment with zinc slightly elevated 
LDH leakage in CT cultures when exposed to 450 umol/L TBH, but nearly doubled 
leakage from TG cells. Treatment with dex/Il-6 enhanced leakage even further in both 
genotypes, but again the increase was greatest in TG cultures. Toxicity was 5-fold greater 
in TG cultures pretreated with dex/Il-6 and exposed to 350 umol/L TBH than identically 
treated CT cultures. Similar to the KO experiment, the combination of zinc treatment and 
dex/Il-6 treatment resulted in TBH-induced toxicity similar to that seen with dex/Il-6 
treatment alone. 



a 

-t-» 

o 
c 



o 

§ 

c 



5000 
4500 
4000 
3500 
3000 
2500 
2000 
1500 
1000 
500 




control 
^ Zn 
§3 Dex/Il-6 
□ Zn + Dex/Il-6 



d 






a a 



KO 



CK 




Figure 5-1. Metallothionein content of metallothionein knockout (KO), knockout control 
(CK), metallothionein transgenic (TG), and transgenic control (CT) mouse hepatocytes. 
Cells were cultured in Williams Medium E (WME) containing either 4 umol zinc/L 
(control), 32 umol Zn/L (Zn), 1 umol dexamethasone/L and 100 units Il-6/mL (Dex/Il-6), 
or 32 umol Zn/L, 1 umol dexamethasone/L and 100 units Il-6/mL (Zn + Dex/Il-6) for 20 
h prior to harvest. MT was quantified for KO hepatocytes cultured in control or zinc 
medium only. Data are means ± SEM (n = 4-8 cultures). Significant differences (p < 
0.05) were determined using ANOVA for a 2x2x2 factorial design, followed by the 
Tukey-Kramer multiple comparison test. 



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Cell viability measured with the MTT assay revealed results similar to 
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Figure 5-2. Tertiary-butyl hydroperoxide-induced cytotoxicity. Cytotoxicity was 
measured as Lactate dehydrogenase leakage from metallothionein knockout (KO), 
knockout control (CK), metallothionein transgenic (TG), and transgenic control (CT) 
mouse hepatocytes after treatment with tertiary-butyl hydroperoxide (TBH). Hepatocytes 
were cultured as in figure 5-1 for 20 h, then treated with 0, 350, or 450 umol/L TBH for 
2 h. (A) LDH leakage from CK and KO mouse hepatocytes. (B) LDH leakage from CT 
and TG mouse hepatocytes. Data are means + SEM (n ■ 5-6 cultures from a 
representative experiment). Significant differences (p < 0.05) between groups treated 
with TBH were determined using ANOVA for a 2x2x2 factorial design, followed by the 
Tukey-Kramer multiple comparison test. 



76 

cultures than CK cultures when exposed to 10 umol/L TBH, and in all KO cultures 
compared to CK cultures at 15 umol/L TBH (Table 5-2). All TG cultures had lower 
viability than CT cultures at 10 umol/L TBH (Table 5-3). Nearly all cells from these 
genotypes were dead at 15 umol/L TBH. 

Total glutathione was measured after the culture period but before the addition of 
TBH to determine if glutathione status was affected by genotype, medium zinc and/or 
dex/H-6 treatment (Table 5-4). Glutathione concentrations were 25-55% lower in CK 
cultures than KO cultures, depending on the culture condition. Dex/Il-6 treatment 
reduced cellular GSH in both genotypes, but to much lower levels in CK cultures. Zinc 
treatment alone, or in combination with dex/Il-6 had little affect on glutathione levels. 

Discussion 

Previous work from this lab found that zinc supplementation protected against 
oxidative stress in primary cultures of rat hepatocytes (Coppen et al. 1988, Schroeder and 
Cousins 1990). Protection was credited, in part, to metallothionein induction by zinc. 
Induction of metallothionein with hormones and cytokines was also correlated with such 
protection (Schroeder and Cousins 1990). We recently found that metallothionein 
expression protected mice against carbon tetrachloride heptotoxicity, but neither 
supplemental dietary zinc nor metallothionein overexpression provided further protection 
in metallothionein knockout and metallothionein transgenic mice (Davis et al. 
submitted). These results were inconsistent with general antioxidant functions for 
metallothionein and supplemental dietary zinc, and are in opposition to a number of 
previously published reports (Liu et al. 1998 A, Liu et al. 1999A, Rofe et al. 1998). With 



77 



Table 5-2. Cell viability after tertiary-butyl hydroperoxide exposure of hepatocyte 
cultures from metallothionein knockout and control mice previously treated with zinc 
and/or dexamethasone and Il-6 abc 





Cell Viability (% of untreated cultures) 


ANOVA Results 


Genotyp* 


i TBH Control 


Zn 


Dex/Il-6 Zn & 






umol/L 




Dex/Il-6 


p value 


CK d 


5 61 ±2 


65 ±2 


63 ± 2 82 ± 1 




KO e 


84 ±1 


70 ±1 


62 ± 2 63 ± 2 
Genotype x Zinc x Dex/Il-6 
Genotype x Zinc 
Genotype x Dex/Il-6 
Zinc x Dex/Il-6 
Genotype 
Zinc 
Dex/Il-6 


0.8090 
0.0001 
0.0001 
0.0001 
0.0625 
0.0741 
0.0655 


CK 


10 54 ±3 


52 ±2 


66 ± 2 72 ± 2 




KO 


90 ±4 


73 ±3 


66 ± 3 62 ± 2 
Genotype x Zinc x Dex/Il-6 
Genotype x Zinc 
Genotype x Dex/Il-6 
Zinc x Dex/Il-6 
Genotype 
Zinc 
Dex/Il-6 


0.4920 
0.0020 
0.0001 
0.0030 
0.0001 
0.0435 
0.8038 


CK 


15 11±1 


9±1 


12±1 18±1 




KO 


32 ±2 


28 ±1 


27 ± 2 26 ± 2 
Genotype x Zinc x Dex/Il-6 


0.5875 








Genotype x Zinc 

Genotype x Dex/Il-6 

Zinc x Dex/Il-6 

Genotype 

Zinc 

Dex/Il-6 


0.0380 
0.0016 
0.0063 
0.0001 
0.7010 
0.5909 



a Cell viability values are means ± SEM, n = 10-11. 
b Zinc treatment was 32 umol/L for 20 h. 

c Dexamethasone and 11-6 treatment (Dex/Il-6) was 1 umol/L Dex and 100 units Il-6/mL 
for 20 h. 

CK, hepatocyte cultures from control mice. 
e KO, hepatocyte cultures from metallothionein knockout mice. 



78 

the experiments described herein we attempted to determine whether metallothionein 
induction and supplemental zinc could act as cellular antioxidants against TBH-induced 
toxicity. 

We used primary hepatocytes cultures from TG and KO mice as a cellular model 
of stably altered metallothionein expression, independent of metallothionein inducing 
agents. In addition we assessed the effects of zinc and/or dexamethasone and IL-6 
treatment on cytotoxicity in these cells, which have inherently different abilities to 
produce metallothionein. In these experiments we found a consistent, direct relationship 
between cellular metallothionein content and susceptibility to TBH toxicity. This 
relationship occurred whether metallothionein was elevated due to genotype, inducing 
agents (zinc or dexamethasone and 11-6 treatment), or their combination. Further, 
dexamethasone and 11-6, powerful metallothionein inducers and protectors against 
hepatocyte toxicity in rats, increased sensitivity to TBH toxicity in these mice. To our 
knowledge, this is the first report that demonstrates an inverse relationship between 
metallothionein expression and susceptibility to an oxidative stress. 

Although the results of this study were unexpected, possible explanations can be 
proposed. Antioxidant defense against the toxicity of tertiary-butyl hydroperoxide 
involves consumption of considerable quantities of glutathione (GSH) by the antioxidant 
enzymes glutathione peroxidase and phospholipid glutathione peroxidase (Rush et al. 
1985). In addition, glutathione transferase enzymes may detoxify genotoxic products of 
lipid peroxidation by conjugatng them to glutathione (Hubatsch et al. 1998). As such, 
disturbances in GSH metabolism may enhance TBH toxicity. For example, hepatocyte 
glutathione levels decline when cysteine availability is low (Wang et al. 1997). In our 



79 



Table 5-3. Cell viability after tertiary-butyl hydroperoxide exposure of hepatocyte 
cultures from metallothionein transgenic and control mice previously treated with zinc 
and/or dexamethasone and Il-6 a ' c 





Cell Viability (% of untreated cultures) 


ANOVA Results 


Genotype 


: TBH Control 


Zn 


Dex/Il-6 Zn & 






umol/L 




Dex/Il-6 


p value 


CT d 


5 82 ±5 


79 ±2 


78 ± 2 80 ± 3 




TG e 


76 ±6 


84 ±5 


94 ± 8 87 ± 5 
Genotype x Zinc x Dex/Il-6 
Genotype x Zinc 
Genotype x Dex/Il-6 
Zinc x Dex/Il-6 
Genotype 
Zinc 
Dex/Il-6 


0.1038 
0.8780 
0.1500 
0.2906 
0.1553 
0.7956 
0.3624 


CT 


10 58±3 X 


48 ± 2 x>y 


53 ± 3 x ' y 48 ± 2 x ' y 




TG 


48 ± l y 


33±2 Z 


29±2 Z 31±l z 
Genotype x Zinc x Dex/Il-6 
Genotype x Zinc 
Genotype x Dex/Il-6 
Zinc x Dex/Il-6 
Genotype 
Zinc 
Dex/Il-6 


0.0415 
0.7802 
0.0054 
0.0007 
0.0001 
0.0001 
0.0001 


CT 


15 18±1 


15±1 


21 ±2 16±1 




TG 


14±1 


13 ±0 


14 ±0 13 ±0 
Genotype x Zinc x Dex/Il-6 
Genotype x Zinc 
Genotype x Dex/Il-6 
Zinc x Dex/U-6 
Genotype 
Zinc 
Dex/Il-6 


0.5971 
0.0204 
0.0434 
0.5886 
0.0001 
0.0021 
0.1061 



a Cell viability values are means ± SEM, n = 9- 1 1 . 

b Zinc treatment was 32 umol/L for 20 h. 

c Dexamethasone and 11-6 treatment (Dex/Il-6) was 1 umol/L Dex and 100 units Il-6/mL 

for 20 h. 

CT, hepatocyte cultures from control mice. 
e TG, hepatocyte cultures from metallothionein transgenic mice. 
x ' y,z Means with different superscript values are significantly different. 



80 

experiments we found that as metallothionein accumulation increased, cellular 
glutathione decreased (Table 4). Since cysteine residues make up 33% of the total amino 
acid content of metallothionein protein, induction of MT protein may consume a 
significant amount of the cellular cysteine pool. Consequently, induction of 
metallothionein by dex/Il-6 treatment may have depleted glutathione levels and enhanced 
TBH toxicity by competing for cysteine needed for GSH synthesis. Further, depletion of 
cysteine pools might have inhibited the GSH regeneration during the toxic insult. 
Alternatively, excess metallothionein may form mixed disulfides with oxidized 
glutathione (GSSG) generated during TBH-induced oxidative stress (Brouwer et al. 1993, 
Chai et al. 1994). Mixed disulfide formation may remove glutathione from its 
regenerative pathway, delaying or preventing regeneration of reduced GSH needed for 
antioxidant protection and other cellular processes (Gilbert 1995, Meister 1995). 

The reduced sensitivity of KO cultures to TBH toxicity might also be explained if 
there is a difference in the peroxidizability of cellular membranes between genotypes. 
For instance, testes of zinc-deficient rats have a lower peroxidizable fatty acid level, and 
are resistant to peroxidation in vitro (Oteiza and Keen 1996). If cells from KO mice act 
similar to zinc-deficient tissues due to the lack of the intracellular ZnMT pool, they may 
also have lower levels of the most peroxidizable fatty acids, and gain resistance to TBH 
toxicity. 

Several cell culture experiments have shown that treatment with metallothionein- 
inducing agents is associated with protection against oxidants (Coppen et al. 1988, 
Schroeder et al. 1990). Similar results were found in cells transfected with 
metallothionein genes (Pitt et al. 1997, Schwarz et al. 1995) or cells from TG and KO 



81 

mice (Lazo et al. 1995, Rofe et al. 1998, Wang et al. 1999, Zheng et al. 1996). Of the two 
studies that used primary hepatocyte cultures from KO mice, one showed only modest 
protection against TBH (Zheng et al. 1996). The other report showed significant 
protection by metallothionein expression against paracetamol toxicity, but only when 
cultures were derived from fed mice; no differences in toxicity were seen between 
genotypes in cultures from fasted mice (Rofe et al. 1998). Further, none of those studies 
investigated whether inducing metallothionein prior to oxidant treatment affects toxicity. 
Several other studies failed to find any protection by metallothionein expression (up to 
166-fold) against the free radicals produced by x-radiation, bleomycin, or doxorubicin 



Table 5-4. Cellular glutathione concentrations in hepatocyte cultures from metallothionein 
knockout and control mice after treatment with zinc or dexamethasone and Il-6 abc 







Cell glutathione (umol/g protein) 


ANOVA 


Genotype 


Control 


Zn Dex/Il-6 Zn & Dex/Il-6 


p value 


CK d 


23 ±3 


24 ± 2 1 1 ± 1 1 1 ± 1 




KO e 


31±3 


40 ± 2 25 ± 3 23 ± 2 








Genotype x Zinc x Dex/Il-6 


0.1066 






Genotype x Zinc 


0.3360 






Genotype x Dex/Il-6 


0.7097 






Zinc x Dex/Il-6 


0.0605 






Genotype 


0.0001 






Zinc 


0.1832 






Dex/Il-6 


0.0001 



a Cellular glutathione values are means ± SEM, n = 6. 

b Zinc treatment was 32 umol/L for 20 h. 

c Dexamethasone and 11-6 treatment (Dex/Il-6) was 1 umol/L Dex and 100 units Il-6/mL 

for 20 h. 

d CK, hepatocyte cultures from control mice. 

e KO, hepatocyte cultures from metallothionein knockout mice. 



82 

treatment (Kaina et al 1990, Kelley et al. 1988). Instead, they found protection to be more 
consistent against alkylating agents. Our results contribute further evidence against a 
cellular free radical scavenging function for metallothionein. 

Supplemental zinc (32 umol/L) did not protect against oxidative damage in these 
experiments. In fact, zinc treatment resulted in a consistent increase in LDH leakage in 
all genotypes (Fig. 5-2). Other studies using mouse hepatocytes also found no protection 
by zinc treatment (50 umol/L and 100 umol/L) against free radical generators (Tezuka et 
al. 1995, Rofe et al. 1998). This is in contrast to several studies using zinc-supplemented 
(48 umol/L) rat hepatocytes (Coppen et al. 1988, Schroeder et al. 1990). Zinc induced 
metallothionein in rat and mouse hepatocytes, but zinc only protected rat cultures. Since 
rat hepatocytes were protected and mouse hepatocytes were not, we cannot rule out 
species difference as a confounding variable in these experiments. For example, mouse 
hepatocytes are significantly more sensitive to TBH-induced toxicity than rats, possibly 
due to a greater peroxidizable lipid content of cellular membranes (Rush et al. 1985). 
Since GSH is an important substrate for detoxification of peroxidized lipids by 
phospholipid glutathione peroxidase and glutathione transferase enzymes, hepatocytes 
from mice may be more sensitive to glutathione depression than hepatocytes from rats if 
lipid peroxidation is greater in mice after TBH treatment. It seems unlikely, however, 
that chemical properties as basic as free radical scavenging (a simple oxidation-reduction 
reaction) and zinc-thiol binding would differ between two rodent species. It should also 
be noted that hepatocytes from two different mouse strains were used in our experiments. 
As such, it is unlikely that the results presented here were due to peculiarities of any 
particular mouse strain. Further, levels of other antioxidants do not differ among these 



83 

mouse genotypes (Iszard et al. 1995, Lazo et al. 1995). Instead, these results show that 
supplemental zinc does not provide consistent protection against oxidative stress, and in 
this case exacerbated toxicity. 

Another interesting result was that dex/Il -6 treatment, which protected rat 
hepatocytes cultures from oxidative stress (Schroeder et al. 1990), enhanced 
susceptibility to TBH in both mouse strains in this experiment. The enhanced 
susceptibility was closely related to the magnitude of metallothionein expressed after that 
treatment, and was not altered by zinc-treatment. This is in opposition to the report of 
Rofe and coworkers (1998), who found that dexamethasone (1 umol/L) protected CK 
mouse hepatocytes against paracetamol toxicity when administered at the same time as 
paracetamol. KO hepatocytes were not protected by dexamethasone treatment, 
suggesting that metallothionein production was necessary for that effect. Metallothionein 
was not induced before paracetamol exposure, however, which may explain why their 
results were different than ours. In support of this, glutathione levels were similar 
between genotypes before paracetamol exposure in their experiments. They also found 
that supplemental zinc nearly tripled metallothionein levels in dexamethasone treated CK 
cultures, yet inhibited dexamethasone-mediated protection against paracetamol (Rofe et 
al. 1998). This suggests that overproduction of metallothionein may be counterproductive 
even when metallothionein induction occurs during paracetamol exposure. 

In conclusion, these results argue that supplemental zinc and/or metallothionein 
do not protect hepatocyte cultures against tertiary-butyl hydroperoxide-induced oxidative 
stress, but rather enhance the toxicity. Further, preinduction of metallothionein enhanced 
the toxicity. Further research is required to determine the mechanism involved, but it is 



84 



likely related to depressed glutathione levels in hepatocytes after metallothionein 
induction. If so, induction of metallothionein prior to oxidant exposure may be 
counterproductive if protection against that oxidant relies on glutathione availability. 



CHAPTER 6 
CONCLUSIONS 



The research reports bound together in this dissertation provide new insights into 
the biochemical and physiological functions of metallothionein and zinc. The foci of this 
research fall into two categories: 

(1) the effects of metallothionein expression and dietary zinc intake in zinc 
absorption, distribution, and intracellular zinc trafficking in TG and KO mice, 

and 

(2) the effects of supplemental zinc, metallothionein expression, and their 
combination in defense against oxidative stress. 

The discussion that follows brings together data from the four separate research 
reports in an effort to provide a unified model of the biological roles of metallothionein 
and zinc, and their interaction. 

Zinc and Metallothionein in Zinc Absorption and Metabolism 

Metallothionein Expression and Zinc Absorption 

The results outlined in Chapter 2 provide further evidence for the theory that 
induction of intestinal metallothionein is a least part of a mechanism for controlling the 
flux of zinc from the intestinal lumen to the general circulation. Specifically, serum zinc 
was elevated to a greater level in mice with lower levels of metallothionein expression 
after an acute oral zinc dose (TG < CT & CK < KO). Similar results were found in a 

85 



86 

separate metallothionein knockout strain over a range from normal to supplemental zinc 
intake (Coyle et al. 1999, Coyle et al. 2000). Contrary to our hypothesis, however, 
intestinal metallothionein induction did not result in greater zinc accumulation in the 
intestine, suggesting that metallothionein 's role is not simply to sequester zinc in the 
intestine. It may be that metallothionein acts to suppress zinc absorption by enhancing 
zinc flux back toward the lumen, as suggested by Hoadley and coworkers (1988). It 
should be stressed, however, that the effects of metallothionein on zinc absorption seen in 
Chapter 2 can be interpreted only with reference to an acute oral dose of zinc. 

Metallothionein Expression and Zinc Metabolism 

The results of Chapters 3 through 5 provide insight into the overall impact of 
metallothionein expression on zinc distribution, accumulation and trafficking at the 
cellular and whole body levels. Data from Chapters 3 and 4 (only h data from Chapter 
4) display the effects of dietary zinc intake (ranging from 10 mg/kg diet to 500 mg/kg 
diet) on metallothionein expression. Conversely, we also saw how the level of 
metallothionein expression affects intestinal, hepatic and serum zinc concentrations. 
Dietary zinc induced intestinal and hepatic metallothionein protein expression, but highly 
supplemental dietary zinc intakes were required (> 200 mg/kg diet). At lower intakes 
metallothionein was undetectable. Further, metallothionein was overexpressed in TG 
mouse liver at all zinc intakes, yet did not affect hepatic zinc accumulation until diets 
containing thirty-fold the requirement were consumed. At 500 mg Zn/kg diet, however, 
hepatic zinc accumulation was directly related to hepatic metallothionein induction in all 
genotypes. These results are directly in line with the role proposed for metallothionein in 
zinc metabolism; i.e., metallothionein is required for zinc accumulation in tissues. 



87 

However, within the range of dietary zinc intakes that we studied, metallothionein 
expression only altered tissue zinc accumulation under conditions of dietary zinc excess. 
The situation might be very different under conditions of stress and zinc deficiency. For 
example, Philcox and coworkers (2000) reported that metallothionein expression inhibits 
intestinal zinc loss during stress (starvation and immune stress associated with 
lipopolysaccharide injection), and prevents body zinc loss during zinc deficiency. 

The results of Chapter 4 also show that hepatic zinc accumulation during stress is 
dependent on metallothionein expression. This had been shown in a separate KO mouse 
model after immune stress (Philcox et al. 1995). Our results extend this finding to the 
level of metallothionein expression observed in TG mice consuming supplemental zinc 
and subsequently treated with carbon tetrachloride. We also report that hepatic 
metallothionein expression after exposure to stress was, in part, dependent upon the level 
of zinc consumed in the diet. In Chapter 5 we report that intracellular zinc accumulation 
in cultured hepatocytes after exposure to excess extracellular zinc, dexamethasone and 
interleukin-6, and their combination was also dependent on the level of metallothionein 
expression. This was also reported recently in another KO mouse model (Coyle et al. 
1995). 

Evidence was found implicating metallothionein in autoreguolation of its own 
gene expression (Chapter 4). Metallothionein expression was stimulated at lower dietary 
zinc intakes in mice with more copies of the metallothionein gene (TG > CT and CK > 
KO). This finding could have larger implications, as it suggests that metallothionein 
expression may also regulate expression of other zinc-responsive genes (Fig. 6-1). At this 
time we speculate that metallothionein acts to regulate gene expression through one of 



88 

two mechanisms: metallothionein may act as a labile zinc pool that can release zinc for 
use in zinc finger transcription factors (e.g., metal response transcription factor-1) that act 
in upregulation of zinc-responsive genes, or metallothionein may directly donate zinc to 
such proteins (Maret 2000, Zeng et al. 1991). Either way, these results suggest that 
metallothionein may be involved in zinc metabolism at dietary zinc levels that do not 
cause detectable changes in tissue zinc accumulation. 

Before leaving this subject, it should be noted that although KO mice have altered 
zinc metabolism, this is only seen under conditions of stress or supplemental zinc intake 
(Chapters 2-5). For example, serum zinc levels are maintained accordingly in KO mice 




Figure 6-1. Autoregulation of metallothionein gene expression. Metallothionein might 
regulate expression of its own gene through one of two pathways: (A) zinc released from 
ZnMT enhances the availability of free zinc for incorporation into transcription factors, 
such as MTF-1, and/or (B) ZnMT directly donates zinc to MTF-1. Either scenario might 
promote the DNA binding activity of MTF-1 by activating a key zinc finger within this 
transcription factor protein. 



89 

allowed to acclimate for several days to diets ranging in zinc content from 10 to 200 
mg/kg. In fact, serum zinc concentrations were lower in KO mice than CK mice 
consuming diets with 300 and 500 mg Zn/kg. Obviously, KO mice can compensate for 
the loss of metallothionein expression over a wide range of zinc intakes. Altered 
expression of one or more zinc transporters may explain this adaptation response in KO 
mice (Liuzzi et al. 2000, McMahon and Cousins 1998). 

Zinc and Metallothionein in Defense Against Oxidative Stress 

In Vivo 

Metallothionein expression protected CK mice against carbon tetrachloride- 
toxicity, confirming metallothionein' s role in protection against oxidative stress. 
Metallothionein overexpression provided no further protection, however, with or without 
added dietary zinc. Supplemental zinc alone was not protective either. These results are 
inconsistent with a general (direct) antioxidant role for either zinc or metallothionein. 
Instead, the result may be more indicative of the general need for rapid zinc accumulation 
in the liver; a process that depends on metallothionein expression (chapters 3-5). We 
speculate that the KO mice are more susceptible to oxidative stress because they lack the 
capacity for rapid and sustained hepatic zinc accumulation. This zinc may be required for 
numerous proteins (enzymes and transcription factors) involved in gene expression, as 
well as other proteins involved in cellular metabolism. Since excess dietary zinc and 
elevated metallothionein expression provide no additional protection, it may be that 
adequate zinc intake and "normal" metallothionein expression are all that are needed to 
maintain these processes. The liver may not be capable of synthesizing proteins quickly 



90 

enough to take advantage of the excess intracellular zinc provided via the diet or elevated 
metallothionein levels. Alternatively, greater production of these proteins may not be 
helpful. 

In Vitro 

Cytotoxicity studies in primary hepatocyte cultures from KO and TG mice were 
undertaken to determine the protective roles of zinc and metallothionein against oxidative 
stress in a simpler, more easily manipulated model. We found that induction of 
metallothionein prior to tertiary-butyl hydroperoxide-treatment resulted in heightened 
cytotoxicity; a result exactly opposite of that hypothesized, and opposite of what had 
been reported previously with other oxidants. This relationship between metallothionein 
expression and cytotoxicity was true whether metallothionein was elevated due to 
genotype, zinc, dexamethasone and interleukin-6, and any combination of these factors. 
Further, the magnitude of cytotoxicity was proportional to the magnitude of 
metallothionein protein induced. 

We found that metallothionein expression was inversely related to cellular 
glutathione levels. Since the first line of defense against the toxicity of tertiary-butyl 
hydroperoxide is the enzyme glutathione peroxidase, which relies on glutathione for 
reducing equivalents, we reason that the depletion of glutathione is responsible for the 
enhanced cytotoxicity observed with metallothionein induction. Not all of the effect of 
dexamethasone and interleukin-6 treatment on glutathione was dependent on 
metallothionein expression, however, since glutathione was also reduced to some extent 
by this treatment in cultures from KO mice as well. 



91 

At least two hypotheses have been generated to explain this phenomenon (Fig. 6- 
2). The first hypothesis (the author's) is that metallothionein induction depletes the 
available cellular cysteine pools. Evidence for this includes the fact that cysteine residues 
make up 1/3 of the total amino acid residues of both metallothionein and glutathione. 
Also, the absolute cysteine content of the metallothionein pool (after induction of 
metallothionein by the various treatments used in this experiment) is comparable to the 
cysteine content of the glutathione pool. Further, the glutathione content of primary 
hepatocyte cultures is highly dependent on the available cysteine content of the culture 
medium (Wang et al. 1997). 

The second hypothesis (of Dr. Cousins) is that the metallothionein induced by 
various treatments physically interacts with glutathione, and thereby interferes with the 



Cysteine 



MT 

4> 



BOH 4 W GSSgBB !^^ NAD 

^GSH Px r w 'GSH Rx 



TBOOH 



NADP 



MT 



Figure 6-2. Mechanisms by which metallothionein might cause glutathione depletion. 
Metallothionein might interfere with glutathione regeneration through physical 
interactions with oxidized glutathione. Alternatively, induction of metallothionein 
might consume enough cysteine so as to interfere with glutathione synthesis. 



92 

use and/or availablility of glutathione as a substrate for glutathione peroxidase. Evidence 
for this viewpoint includes the fact that glutathione forms mixed disulfides with a number 
of intracellular sulfhydryl-containing proteins, as well as the fact that several putative 
glutathione binding sites have been reported in the metallothionein molecule (Brouwer et 
al. 1993, Maret 2000). This hypothesis includes observations from in vitro experiments 
where glutathione peroxidase and reduced glutathione release zinc from metallothionein 
and cellular oxidants cause release of cellular glutathione (oxidized and reduced). There 
is no reason to believe that the mechanisms outlined in these hypotheses would be 
mutually exclusive. If metallothionein does affect hepatic glutathione levels in vivo, this 
relationship might explain hepatic glutathione depression seen after treatment with a 
certain hormones, or under some stress conditions. 

In summary, the findings of these studies agree with the previously proposed 
theories regarding the role of metallothionein in zinc metabolism. The results herein 
refine this model by constraining it to conditions of highly excessive zinc intake. A more 
novel finding was that metallothionein may regulate its own expression by providing a 
labile intracellular zinc pool that may feedback positively on metallothionein gene 
expression. 

Metallothionein expression protected against oxidative stress in vivo, but 
metallothionein overexpression and supplemental dietary zinc provided no further 
protection. We interpret this to mean that there is a threshold of zinc intake and 
metallothionein expression that is protective, but above which is not helpful. Normal 
metallothionein expression combined with the required zinc intake meet this threshold 
value in mice. These results are not consistent with direct antioxidant roles for 






93 

metallothionein or zinc. The results may be more reflective of the need for appropriate 
regulation of zinc metabolism during oxidative stress, since zinc metabolism was highly 
altered in KO mice. Further, studies in primary hepatocyte cultures suggest that 
glutathione depletion may result in cells that experience metallothionein overexpression, 
and that glutathione depletion may compromise oxidant defenses. 






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BIOGRAPHICAL SKETCH 

Steven Roger Davis was born on August 3, 1968 in Worcester, Massachusetts. He 
was raised in Sutton, Massachusetts and graduated from Sutton High School in 1986. He 
received his Bachelor of Science degree in Chemistry at Worcester State College in 
Worcester, Massachusetts in 1992. He then worked as a chemistry laboratory 
instructor/supervisor for two years at the College of the Holy Cross in Worcester, 
Massachusetts before coming to the University of Florida to pursue a doctorate in 
Nutritional Sciences. 









109 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quantity, as a dissertation for the degree of Doctor of Philosophy 




Robert J. Cousins, Chair 
Boston Family Professor of 
Human Nutrition 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quantity, as a dissertation for the degree of Doctor of Philosophy. 

5esse F. Gregory, III \U -J 
Professor of Food Science and 
Human Nutrition 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quantity, as a dissertation for the degree of Doctor of Philosophy. 

Susan S. Percival 

Associate Professor of Food Science 
and Human Nutrition 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quantity, as a dissertation for the degree of Doctor of Philosophy 




*:*zLS 



Rachel B. Shireman 
Professor of Food Science and 
Human Nutrition 

I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and 
quantity, as a dissertation for the degree of Doctor of Philosophy. 




Stephen M. Roberts 

Professor of Veterinary Medicine 



This dissertation was submitted to the Graduate Faculty of the College of Agricultural 
and Life Sciences and to the Graduate School and was accepted as partial fulfillment of 
the requirements for the degree of Doctor of Philosophy. 



December 2000 T^' 



«srQ_ 





Dean, College of Agricultural an 
Life Sciences 



Dean, Graduate School 












LD 
1780 

20 M 

UNIVERSITY OF FLORIDA 



3 1262 08555 3658